Molecular Markers: Indispensable Tools for Genetic Diversity Analysis and Crop Improvement Biotechnology

Molecular Markers: Indispensable Tools for Genetic Diversity Analysis and Crop Improvement Biotechnology
Molecular Markers: Indispensable Tools for Genetic Diversity
Analysis and Crop Improvement Biotechnology
*1Md. Anowar Hossain, 2Md. Sajjad Hossen, 3Md. Rezaul Karim
1,2,3Department of Biochemistry & Molecular Biology, University of Rajshahi, Rajshahi-6205, Bangladesh
Recent progress in molecular biology has led to the development of new molecular tools that
offer the promise of making plant breeding faster. Molecular markers are segments of DNA
associated with agronomically important traits and can be used by plant breeders as selection
tools. Breeders can use marker-assisted selection (MAS) to bypass the traditional phenotype-
based selection methods in order to improve crop varieties with pyramiding the desirable traits
within short time. Various molecular markers such as RAPD, SSR, ISSR, RFLP, AFLP, SNP, SCAR,
CAPS, etc. are extensively used for plant genetic diversity studies and crop improvement
biotechnology. These markers are different in characteristic properties, applicability to various
plants, unique in the resolving power and also have own advantages and disadvantages. This
review article provides a valuable insight into different molecular marker techniques,
classification, their advantages, disadvantages, ways of actions, uses of molecular markers in
plant genetic diversity analysis and quantitative trait loci (QTL) mapping. It could be helpful for
plant scientists and breeders in MAS breeding and crop improvement biotechnology in the post-
genomic era.
Key words: Molecular markers, Genetic diversity, Marker assisted selection, Quantitative trait loci, Biochemical markers.
INTRODUCTION
DNA (Deoxy Ribonucleic Acid) is the genetic material that
distinguishes one organism from another at molecular
level. DNA is packaged in the chromosome in pairwise
manner where each chromosome comes from parent
genome. The genes are segments of the DNA that are
responsible for carrying specific characteristics located on
specific sites in the chromosomes. All of the genes reside
in a single gamete is simply called genome (Semagn et al.,
2006). The full genome sequence is available only for
some plant such as Arabidopsis, Rice, wheat, tomato,
maize, barely and other economically important crops
(Semagn et al., 2006). Therefore, it is very difficult for
scientists to analyze the genetic variability of specific plant
species without the genome sequences. For identifying
specific genes in un-sequenced plants, located in the
chromosome most scientists use a special tool namely
genetic marker (Jonah et al., 2011). A genetic marker can
be described as a segment that has a specific location on
the chromosome and is associated with a particular trait or
phenotypic character. Or it can be described as a variation
caused by mutation or observed changes in the genomic
loci (Al-Samarai and Al-Kazaz, 2015). A molecular marker
can also be defined as a sequence of DNA, located on a
known position on the chromosome, or it can be classified
as a gene whose phenotypic expression is frequently
easily observed and used mainly to detect an individual, or
as a probe to identify chromosome, nucleus, or locus
(Collard et al., 2005). A molecular marker can identify the
difference between particular genomes. Molecular marker
techniques are designed based on polymorphisms that
occur naturally in DNA sequences (Westermeier, 1993). It
sometime does not correlate with phenotypic characters.
A molecular marker can be considered as a constant
landmark of the genome as they don’t have any known
biological function and they are transferred from one
individual to the other following the Mendel's law of
segregation (Kumar, 1999). They work by detecting
*Corresponding Author: Dr. Md. Anowar Hossain;
Department of Biochemistry & Molecular Biology,
University of Rajshahi, Rajshahi-6205, Bangladesh.
Email: mahossain95@hotmail.com
Co-Author Email: 2
sajjadbmbru@gmail.com,
3
drmrkarim@yahoo.com
Review Article
Vol. 7(1), pp. 613-623, January, 2020. © www.premierpublishers.org, ISSN: 2167-0449
International Journal of Plant Breeding and Crop Science

Hossain et al. 614
differences of DNA sequences between individual’s
genotype. Molecular markers have several advantages
over conventional marker system as they are stable and
detectable in all the plant tissues regardless of growth,
development and any other physiological state and usually
it is not influenced by the epistatic and environmental state
of the plants (Waseem et al., 2012).
The idea of genetic marker does not come suddenly. In the
19th century, Mendel used phenotype based molecular
marker system (Waseem et al., 2012). Later the results of
phenotype-based marker of Drosophila melanogaster
played an important role to establish the genetic linkage
theory (Jonah et al., 2011; Kumar, 1999). Since then
morphological characters were used as marker system for
the identification of specific character. Cytological markers
were used extensively in the past but the unusual
occurrences and extremely laborious process make this
marker unpopular (Kumar, 1999). Biochemical and other
protein-based markers are considered as the first set of
molecular marker system that are used mainly for the
detection of the variance of amino acids. DNA markers
detect the variance of the DNA sequences of individuals
and these markers are not changeable with the
environmental effects. They function depending on the
DNA sequences in contrasts to cytological and
biochemical markers that rely on visible characters and
traits and protein produced by genes respectively (Al-
Samarai and Al-Kazaz, 2015). In the post-genomic era,
molecular marker development is being much easier using
next-generation sequencing of the whole genome and
transcriptome sequencing technique. This review article
deals with the basic principles of molecular markers, their
ranges, advantages and disadvantages in the genetic
diversity studies, linkage mappings and marker-assisted
selection for crop genetic improvement and future
direction.
Basic characteristics of molecular markers
Various categories of molecular markers are used in the
diversity analysis. They vary in their characteristics and
properties. However, some of the ideal characteristics of
the molecular marker are illustrated here. According to the
recent published reports (Semagn et al., 2006; Millee et
al., 2008; Mondini et al., 2009), an ideal molecular marker
should possess the following criteria. Molecular marker
should be reproducible. Reproducibility means that the
marker when used it will give the same result in every time.
Molecular marker should be highly polymorphic. It should
be easily accessible without cloning or sequencing.
Sometimes developmental cost for some markers
becomes so high to be affordable. Markers must have an
easy exchange of data between different laboratories for
collaborative research work. An ideal molecular marker
has co-dominant inheritance and has selective neutral
behaviour with no pleiotropic effect. The experimental
assay should be easy and rapid. Development of the
markers should be at reasonable cost and occurrence at
the genome should be very frequent.
It is very difficult to find a molecular marker with all the
above characters. Therefore, a particular molecular
marker can be selected for a particular study based on
some important properties like the expected level of
variation, time allowances of a specific project, extend of
genetic diversity information, level of operation and
expense of different molecular markers (Karp et al., 1997).
It is also important to estimate whether a single marker
system can cover all the queries of a particular project.
Otherwise, combination of two or more molecular markers
can be used to answer all the questions (David and Arvind,
2006). Nowadays, various types of molecular marker are
used to estimate genetic diversity of plants classified as
the hybridization-based molecular marker and the
Polymerase Chain Reaction (PCR) based molecular
marker (Semagn et al., 2006). In the hybridization-based
molecular marker system DNA sequences are visualized
by hybridizing the restriction endonuclease digested DNA
with a labelled probe that is a DNA of known sequences.
In PCR based molecular marker system DNA fragments
are amplified in the thermal cycler using specific or
arbitrary sequences (primer). Some primers are gene-
specific and some primers are randomly selected. The
amplified PCR products are visualized by electrophoresis
or autoradiography. After electrophoresis, the products are
visualized under the gel documentation system. PCR is
extremely sensitive and functions very rapidly.
Hybridization-based molecular marker system is time-
consuming and complicated in their principle. For this
reason, PCR based molecular marker is used very widely
in recent years.
Genomic DNA isolation is the first step to work with
molecular markers
Extraction of genomic DNA (Mitochondrial, Nuclear or
Chloroplasts) from the targeted species is the initial stage
of all the molecular marker system. DNA may be extracted
using fresh, lyophilized or from a dry sample. Fresh young
leaves of plants are used to extract best quality DNA.
There are different methods of DNA extraction, some
methods are highly complicated and some are simple and
rapid and it also varies with the quantity and the quality of
the extracted DNA (Naqvi, 2007; Ikeda et al., 2001; Von
post et al., 2003). There are also some laborious and time-
consuming protocols that can yield good quality DNA
(Murray and Thompson, 1980; Dellaporta et al., 1983).
DNA extraction in its initial step requires breakings or
digesting the cell walls for realizing the cell materials. Lipid
materials of the cells are removed by using detergents like
SDS (Sodium Dodecyl Sulphate), CTAB (CetylTrimethyl
Ammonium Bromide), and MTAB (Mixed Alkyl Ammonium
Bromide). CTAB method is widely used and is effective for
high-quality DNA extraction from plant sources. CTAB
buffer contains EDTA that is required for chelating the
magnesium ion that is a very important cofactor. For the
extraction of good quality DNA, it is important to remove
the polysaccharide, phenolics, carbohydrates and other
impurities. NaCl is used with the CTAB buffer that removes

Int. J. Plant Breed. Crop Sci. 615
the major polysaccharides and phenolics (Paterson et al.,
1993). Some of the protocols used KCl in place of NaCl
(Thomson and Henry, 1995). The protein contaminants of
the DNA are denatured from the ground sample by using
protein degrading enzyme or the samples are heated at
65°C with the Extraction buffer. The extracted DNA also
contains a huge amount of RNA that is removed by an
RNA degrading enzyme called RNase A.
After treatment with the buffer, it needs to separate the
degraded materials such as RNA, proteins,
polysaccharides, and phenolics from the extracted DNA,
which is done by centrifugation. An aqueous phase, which
contains genomic DNA, is then transferred to another tube
containing salts (Sodium acetate) for precipitation. Finally,
washing the DNA with the wash buffer (absolute ethanol
can also be used). Extracted DNA is then dissolved in
highly purified water or buffer. Qualitative and quantities
measurement of the extracted DNA can be done by either
electrophoresis or by spectrophotometry. Generally, gel
electrophoresis determines whether the DNA has any
degradation. DNA concentration is measured at 260 nm
and quality is checked by taking the absorbance at
260/280 ratio. For the molecular marker based genetic
diversity analysis, it is very important to extract high quality
DNA having no contaminations or PCR inhibitors. Since
biochemical parameters of different plant species are
variable, case-by-case modification of DNA extraction
protocols are required to achieve good quality DNA.
Classification of Molecular Markers
i) Non-PCR based molecular marker:
Restriction Fragment Length Polymorphism:
Restriction Fragment Length Polymorphism (RFLP) is the
first and the most widely used Hybridization based DNA
marker and was first used in the detection of DNA
sequence polymorphism for gene mapping of a
temperature sensitive adenovirus serotype (Grodzicker et
al., 1975; Millee et al., 2008). Basically, it was used to
investigate the relationship among very closely related
taxa (Miller and Tanksley, 1990). Molecular marker acts as
a fingerprinting tool for study of genetic diversity and
hybridization as well as assessing the gene flows between
crops and weeds (Desplanque et al., 1999). This
molecular marker system is based on the pattern
difference capability of the restriction enzyme. The basis
of RFLP is to detect the nucleotide mutation like insertion,
deletion, substitution, inversion in the whole genome that
can create new restriction site (Yang et al., 2013). Genetic
information is stored in the DNA sequences within the
genotype and the basis of genetic variation is the
differences of this DNA sequence. Plants and animals are
able to replicate these sequences with high accuracy and
very rapidly but sometimes DNA sequences are changed
by different physiological pathways or environmental
factors. These changes can cause by insertion, deletion,
translocation, transposition, inversion or duplication of the
sequences and these differences in the sequence may
sometime cause the gain or loss of a fragment at the
restriction sites.
Restriction fragments of different lengths can be
recognized by using southern blot and labelled probes.
Though RFLP markers are not used widely it has some
importance in the identification of genetic diseases or to
identify a person who is prone to disease and also are
used for the detection of the carrier for genetic disease
(Emadi et al., 2010). The procedures for RFLP includes:
a. Digesting genomic DNA with a restriction enzyme.
b. Agarose gel electrophoresis for the separation of
restriction fragments.
c. Restriction fragments then blotted with a membrane
and detected with a labelled probe.
d. Autoradiography
RFLP markers are highly reproducible and co-dominant
inheritance. These markers are locus-specific and can be
easily transferred between different laboratories. It is very
easy to detect differences between sequences due to
large differences of the fragment size and no sequencing
of the genome is required for diversity study. However,
RFLP has some limitations. It requires a very large amount
of DNA being highly purified, high molecular weight
(Young et al., 1992). It has low level of polymorphism and
low amount of restriction fragments are detected per
assay. It is also time-consuming, laborious and expensive
process. The major concern is that it uses radioactive
probes. This marker system relies on the development of
a specific probe for each organism.
ii) PCR-based Molecular Marker:
Polymerase Chain Reaction (PCR) is a molecular biology
technique that involves in vitro uses of thermostable DNA
polymerase to amplify specific fragments of DNA without
using the living organism (Yang et al., 2013). Kary Mullis
invented PCR in 1983 and since then various PCR based
molecular marker techniques have been developed. In the
PCR reaction DNA sequence of the targeted fragment are
amplified using a series of heat cycles of repeated
denaturation of the DNA sequences, primer annealing for
the primer to bind to its appropriate sequence and
extension for DNA polymerase to produce new nucleotides
complementary to the existing bases. Each amplification
cycle has the potential to double the amount of DNA from
the previous cycle thus having an exponential growth of
the nucleotide bases (Bertlett and Stirling, 2003). There
are some differences of DNA sequences between two
individuals of the same species. For the difference in the
DNA sequences, one fragment can produce in one
individual while not in the other individual. These
differences in the DNA fragments can be used in the
molecular fingerprinting or diversity analysis between
different individual (Welsh and McClelland, 1990). Taq

Hossain et al. 616
DNA polymerase used in PCR amplification was isolated
from the bacterium Thermus aquaticas. PCR based
molecular marker has several advantages over the
hybridization based molecular marker system such as-
a. Less amount of DNA is required than hybridization
based molecular marker.
b. No radioisotope is used.
c. Higher polymorphism, highly reproducible and more
reliable.
d. It requires less time as compared to hybridization-
based markers.
e. No prior information is required for the DNA
sequences of interest.
f. Less expensive and markers are easily available.
There are two types of PCR based molecular markers
depending on the primer used for amplification of the DNA.
a. Arbitrary or semi-arbitrary primed sequence that does
not require pre-sequencing DNA information.
b. Site-tagged DNA sequences that are developed using
known sequences.
Random Amplified Polymorphic DNA (RAPD)
RAPD pronounced as Rapid is known as the firstly
developed PCR based molecular marker system
(Crossland et al., 1993). In this technique, short
oligonucleotide sequences (10 bases long) are utilized for
the amplification of nanogram amount of genomic DNA
under low annealing temperature by PCR. Decamer
primers are readily available from different companies
(Sen et al., 2010). RAPD primers bind at a different binding
site of the DNA but it is not certain exactly where the primer
binds. This property makes the RAPD popular for
analyzing genetic diversity of unstudied species of which
sequence data are unavailable Amplified fragments are
detected and used for the genetic diversity study. The
amplified fragments size ranges are usually of 0.2.5-5 kb
and separated by agarose gel electrophoresis for the
detection of polymorphism. RAPD marker is now widely
used all over the world for the identification of accessions,
plant breeding and genetic diversity analysis (Fukuoka et
al., 1992).
Using RAPD markers for genetic fingerprinting within
different species of a closely related group is quite
beneficial than any other markers for its simplicity and
availability to all over the world (Gupta et al., 2001; Weising
et al., 1995). It requires a very less amount of DNA to work
with the genome that is not possible with RFLP. It is quite
a simple and quick process. RAPD is less technology
intensive, cheaper and easily available than any other
molecular markers. It does not require any prior knowledge
of the targeted species for designing primers (Weising et
al., 2005; Edwards and McCouch, 2007). Working with this
marker does not have any involvement of radioactive
probe and hybridization or blotting is not required for
assessment of the genetic variability.
However, RAPD markers also have some limitations, the
major one is that RAPD marker lakes of producing
reproducible results. These primers are very short and
single primer used for amplification. Single mismatch of a
nucleotide can inhibit from producing the band. RAPD
markers sometimes show co-dominant inheritance. This
marker is not locus-specific so banding pattern can’t be
interpreted in terms of locus and alleles.
Amplified Fragment Length Polymorphism
Amplified fragment length polymorphism (AFLP) is a DNA
fingerprinting technique, which was introduced by a Dutch
seed company KeyGene to overcome the reproducibility
problem of RAPD marker (Vos et al., 1995). AFLP marker
has the widest application in the genetic diversity and in
the study of population structure and differentiation. This
molecular marker method is based on the PCR
amplification of the selected restriction digested fragments
of the full genome. This marker technique combines the
power of RFLP and RAPD to ligate fragments of DNA
regardless of its source and without any pre-sequencing of
its genome sequence and the analysis of AFLP is also very
easy because polymorphism is identified by observing
absence or presence of bands rather than mapping the
size of different locus (Mondini et al., 2009). AFLP is an
extremely sensitive method and the use of the fluorescent
primer for automatic fragment detection and the software
packages to analyze the fragments makes it really
convenient for use in the population diversity analysis.
Generally, 75-150 fragments are generated for each
primer combination and each represents a unique primer
binding site (Farooq and Azam, 2002). AFLP markers are
popular all over the world for use in population genetics,
systematic, pathotyping and Quantitative Trait Loci (QTL)
mapping (Mueller and Wolfenbarger, 1999).
Major advantage of AFLP marker is that no prior
knowledge of the targeted DNA is required for AFLP
analysis (Saal and Wricke, 2002). It is reproducible and
highly reliable than RAPD. Analysis by AFLP is rich in
information as a lot of fragments are produced by a single
combination of primer than the RAPD, RFLP, and
microsatellites. As PCR is very fast and high multiplex ratio
makes the AFLP technique highly suitable. However, this
method is time-consuming than other types of molecular
marker method. It requires highly purified DNA sample
which must be free of restriction enzyme and any other
types of PCR inhibitor. As like as the RAPD the fragment
produced by the RFLP are dominant and thus it is not
preferable to distinguish between homologous and
heterologous genome. RFLP requires to purchase
restriction enzymes, ligation enzymes and the adapters
which makes this process costly.
Simple Sequence Repeat
Microsatellite or Simple Sequence Repeats (SSR) also
known as Variable Number of Tandem Repeat Sequence

(VNTR) and Simple Sequence Length Polymorphism
(SSLP). These are widely dispersed in the eukaryotic
genome and variation in the tandem repeat sequence of
the core sequence occurs by the polymorphism of these
specific locations (Wan et al., 2004). Microsatellites are
short stretch of DNA of which one to six bases can repeat
over five to hundred times at each locus (Litt and Luty,
1989). Microsatellites occur not only in the nuclear
genome but also in the mitochondrial and chloroplast
genome as a repetition of guanine and cytosine (Jarne and
Lagoda, 1996). It is possible to isolate microsatellite from
any target species as one hundred five microsatellite loci
are present in the genome. Microsatellites are found in the
genome as co-dominant Mendelian pattern and can reveal
as homozygote and heterozygote in each individual. This
marker is used to identify structure, classification,
discrimination, the relationship in individual and population
(Jarne and Lagoda, 1996).
Microsatellite markers are highly polymorphic they can be
used to compare genomes of two closely related groups.
It requires a very small amount of DNA for the experiment
and can be easily automated by high throughput
screening. Microsatellite markers are more information
rich and more variable than RAPD, AFLP, and RFLP. The
results can be obtained by visualizing the amplified
product in agarose gel electrophoresis (Matsuoka et al.,
2002). They are a co-dominant marker and preferred for
genetic mapping and population genetics. Microsatellites
are widely distributed within the different population. This
marker is considered as the best marker for detection of
inter-varietal diversity and polymorphism. Use of
fluorescent markers makes it highly reliable and high
throughput analysis.
One of the major constraints for SSR markers is the high
cost for the development of SSR marker for a specific
species. It is also very laborious and time-consuming.
These markers are gene-specific and prior knowledge of
the target species of interested is a must. This makes SSR
so difficult to analyze genetic variation of a genetically
unstudied group. Because of the differences in SSR allele,
resolution of the amplified product is better in
polyacrylamide gel than in agarose gel. The cost of
polyacrylamide gel is higher than agarose gel. It is not
always possible of exchange of information between
different laboratories because of the inconsistencies of the
SSR allele size.
Inter-Simple Sequence Repeat
Inter-Simple Sequence Repeats (ISSR) are 100-3000 bp
long fragments adjacent between two identical
microsatellite regions in the opposite orientation
(Zietkiewicz et al., 1994). It uses the single microsatellite
primer to amplify different sized inter simple sequence.
ISSR markers are very simple and randomly distributed in
the genome and use of radioactivity is not essential for this
marker. They require a very low amount of DNA and
exhibit a dominant inheritance pattern. The primers used
in ISSR are usually longer (15-30 bases) which give it
higher annealing temperature and higher strength (Joshi
et al., 2000). The amplified PCR product is generally 200-
2000 bp long and can be viewed on both agarose gel and
polyacrylamide gel through silver staining. Though ISSR
has the characteristics of microsatellite it does not require
any prior knowledge of genome of interest. ISSR markers
usually are used extensively in the taxonomic
identification, gene mapping study, identification of strain,
genetic identification and diversity analysis. ISSR markers
are also used for gene mapping, identification of variables,
genetic diversity analysis, taxonomy etc. (Nilkanta et al.,
2017).
ISSR analysis does not require any pre-sequencing of the
species of interest. Thus, it can use the facilities of random
primer. This molecular marker technique is quick, less
technology intensive and inexpensive. Their polymorphism
rate is very high among closely related species. Very small
amount of DNA is required for the experiment. ISSR
fragments are distributed randomly throughout the whole
genome and can be easily isolated. Like the RAPD, ISSR
also have reproducibility problem. Sometimes the level of
polymorphism varies with the detection method. It usually
shows better polymorphism in polyacrylamide gel than in
the agarose gel. Co-migration of the fragments from the
non-homologous genome is another limitation of ISSR.
Single Nucleotide Polymorphism
Single Nucleotide Polymorphism (SNP) is the most
commonly found genetic variation between two individuals
and most abundant variation in the genome that occurs
within 1 nucleotide among 1000 bases. It has recently
emerged as a new generation molecular marker coming
after RFLP and SSR (Nilkanta et al., 2017) for various
applications in the assessment of plant genetic variation.
They are an attractive and essential tool for gene mapping,
marker assisted breeding of crops, and map-based cloning
of genomes (Yu et al., 1994). SNP occurs when a single
nucleotide between two genomes is changed. SNP
genetic variation can be occurred by transversion,
transition, insertion and deletion. Mutation by transition is
the most frequent of all the SNP type. In the genome, the
occurrence of the SNP is mainly in the noncoding region.
Sometimes it also occurs in the coding region of the
genome, which results in the formation of a non-
symptomatic mutation in the amino acid sequence or can
generate an asymptomatic mutation that does not change
amino acid sequence. As it consists of co-dominant and
binary characteristics, SNP can efficiently differentiate
between heterozygous and homozygous characters of
alleles (Arif et al., 2010). In plants, SNP can be designated
by a method called Diversity Array Technology (DArT)
from the EST and single nucleotide pyrosequencing
(Zhang et al., 2017; Jiménez-Gómez and Maloof, 2009).

Hossain et al. 618
SNP markers have several advantages over the other
molecular markers. They are genetically abundant,
ubiquitous and stable than other molecular marker type
and flexible for high throughput analysis (Vignal et al.,
2002; Mammadov et al., 2012). For the studies of multi-
factorial disease, the use of SNP marker has greatly
increased (Jarne and Lagoda, 1996). SNP has proved to
be a universal marker for assessment of genetic variation
among individuals of the same species (Mammadov et al.,
2012). On the other hand, most important limitation of SNP
marker is that it contains low level of information as
compared to the microsatellite marker and this problem
can be solved by using whole genome sequencing
(Werner et al., 2002; Werner et al., 2004).
Cleaved Amplified Polymorphic Sequences
Cleaved Amplified Polymorphic Sequences (CAPS) are
the combination of RFLP and PCR (Idrees and Irshad,
2014). CAPS are DNA fragments amplified by PCR using
a 20-25 base length primer followed by digesting the DNA
with restriction enzymes (Baumbusch et al., 2001). Assay
of CAPS marker depends on the variation in the length of
fragments caused by the differences in the nucleotide
sequences of different samples. The variations in
restriction enzyme-digested fragments are identified by
resolving in the gel electrophoresis. As it combines the
PCR and RFLP, CAPS are called PCR Restriction
Fragment Length Polymorphism (PCR-RFLP).
CAPS have several advantages over RFLP. As CAPS
involves the PCR amplification in its assay, it requires a
very low amount of DNA per reaction (50-100 ng per
reaction). CAPS markers are co-dominant and highly
reproducible (Matsumoto and Tsumura, 2004). It does not
require the laborious and time requiring steps of
hybridization with labelled probe making this marker more
suitable. In the genetic mapping, CAPS marker derived
from EST are more potential than the markers developed
from non-functional sequences such as the microsatellite
markers. The limitation of CAPS is that the detection of
polymorphism in the samples are more difficult than the
RFLP analysis because of the limited size of the fragments
and sequence data is also required for analysis.
Sequence Characterized Amplified Region
Michelmore et al., (1991) firstly introduced Sequence
Characterized Amplified Region (SCAR) molecular marker
technique. This marker is a special type of molecular
marker in which the RAPD amplified fragments are
sequenced and amplified again by longer primers (22-24
bases). Specific fragments of interest from the RAPD
amplified DNA are cloned and sequenced and SCAR
markers are developed from the sequence information of
the RAPD fragment and following by PCR amplification in
more stringent condition (Shen et al., 2011). In the
conversion of SCAR from RAPD, a co-dominant marker
obtains an additional advantage in the analysis of genetic
diversity although this marker sometimes shows
dominance when both of the primers overlap with the
sequence variation site. As SCAR markers are locus
specific, it successfully applied in the gene mapping and
mostly in the marker assisted selection (Paran and
Michelmore, 1993). Main advantage of SCAR marker is
that it overcomes the low reproducibility problem of RAPD.
The assay is also very rapid and quick. Very negligible
amount of DNA (50-100 ng per reaction) is required for
PCR amplification (Menezes et al 2002). SCAR markers
are usually used to species-specific identification of
threatened organism (Roslan et al., 2017). The only
drawback of SCAR marker is that it requires DNA
sequencing for analysis of the fragments (Ren et al.,
2012).
iii) Functional Molecular Markers:
Advancements in the NGS technologies in conjugation
with different tools of molecular biology including
metabolomics, proteomics, genomics and associated
mapping study facilitate the detection of candidate gene
and identification of their allelic variants. Integration of
marker system and these tools enhanced the crop
improvement program by using functional molecular
markers (FMMs). These FMMs are designed from the
sequence variation among functional genes, which are
associated with phenotypic variation. Functional molecular
markers excluded of the problems related to random DNA
markers and for this reason FMMs are also known as ‘The
holy grail’ of plant breeders who employ targeted MAS
breeding for crop improvement. The FMMs have been
developed and used for the improvement of cereal crops
for agronomic, food quality, disease resistance and abiotic
stress tolerance traits (Kage et al., 2016).
Uses, advantages and shortcomings of molecular
markers
Different marker systems are used in molecular biology for
diversity analysis depending on the lab facilities and other
criteria of the molecular markers. Specific markers are
used for specific purposes; therefore, selection of
appropriate marker is one of the major tasks for genetic
diversity assessment. DNA and PCR based molecular
markers are used extensively in recent years as they have
many advantages and are biased by environmental
factors. Shortly we can compare different molecular
markers on some specific criteria.
i) Genomic abundances
The total number of markers produced depends on the
specific sites from where it generates within the genome.
In the eukaryotic genomes, SSR marker occurs frequently.
RAPD, RFLP, AFLP markers also occur very frequently in
the genome for a large number of restriction sites in the
genome (Table 1). RAPD markers are most common
because of numerous random sequences are used for this

marker. In addition, some primer such as CAPS, SCAR,
SSR markers require sequence data of the sites are
required for primer construction which limits the use of this
kind of markers for some specific crops. Genomic
abundances of the molecular marker study are also
important factor to identify which fraction of the genome
needs to be taken for constructing the linkage maps.
Allozymes and other protein based molecular markers are
not as frequent in the genome because of low detection
capability of the markers.
ii) Reproducibility
Reproducibility means the production of identical data in
different laboratories. This is very important for
collaborative research work and very important criteria for
molecular markers. To obtain reproducible data it is very
important to very sharp and highly purified DNA.
Fragmented or smeared DNA does not give reproducible
results. Reproducibility by RAPD marker is rare even when
highly purified DNA is used because of the shortness of
the length of the primer (10 base). These short primers
often interfere with the annealing temperature of the DNA
samples resulting in the deviation from the standard
results. For this reason, standard PCR condition is used
for RAPD analysis. This problem can be overcome by
using mapped marker with longer primers than the random
primers.
iii) Labour intensive
PCR based molecular marker systems are less laborious
and are automated process but still the DNA sequencing
based molecular markers may be labour intensive.
Hybridization-based markers like RFLP and AFLP markers
include time-consuming steps like southern blotting,
hybridization, labelling with probes, which are highly
laborious procedure and require skilled person. Automated
sequencing method has greatly reduced time requirement.
PCR based markers like RAPD, SSR ISSR are less
laborious methods.
iv) Development cost
Development cost for a marker is an important criterion for
selecting an appropriate marker system for a specific
function. Site-specific molecular markers like SSR, SCAR
and SNP are required for the development of markers from
the gene of interest. This makes this marker system highly
expensive for developing the primers. Another
hybridization-based marker like RFLP AFLP requires
cDNA library construction and examinations of the various
restriction enzymes to detect the polymorphism. Random
primers like RAPD does not require primer development
and hence are not so expensive (Table 1).
v) Operation Cost
Technical apparatus, consumables, reagents used all are
included in the operational cost. All PCR based markers
require a common expensive reagent, Taq polymerase
with buffers and the hybridization based marker like RFLP
requires restriction enzymes and the polymorphism
detecting system (Autoradiography). RAPD, SSR, SCAR
markers use agarose gel electrophoresis staining with
Ethidium Bromide that are much cheaper than the
polyacrylamide gel electrophoresis with silver staining or
autoradiography for detection of bands. The automated
method of PCR for different markers is also very expensive
and also requires technically skilled persons. RAPD
markers are less technology intensive and are
comparatively less expensive. Generally, the operation
cost of different markers varies with the methods they
used. Automated marker system equipments are very
expensive and also very sensitive (Table 1). However,
high-operational cost could be reduced by multiplexing or
outsourcing of the samples to other laboratories.
vi) Quantity of template DNA required
RFLP and Microsatellites require large quantity of DNA for
a reaction to perform (5-10 µg). Southern blot needs to be
probed for several times. The PCR based molecular
markers use the lowest amount of DNA for a single
reaction (50-100 ng) and are always preferred for this
reason. Medium quantity of DNA (0.3-1µg) is required for
AFLP analysis because of the restriction of the DNA prior
to the PCR reaction. If a very little amount of DNA obtained
from a particular species, PCR based markers are always
preferred.
vii) Co-dominant or dominant characteristics
Co-dominant markers usually expressed both the alleles
in an individual. Therefore, with the help of co-dominant
marker homozygotes and heterozygotes genes can be
differentiated. In contrast, the dominant markers are multi-
locus markers and amplified bands are analyzed by
calculating the absence or presence for a particular locus
and dominant marker does not allow differentiation of
homozygotes and heterozygotes. Co-dominant marker is
a single locus marker, which is always preferred.
Information gained from the co-dominant markers is lower
than the multi-locus markers. SNP, microsatellite markers
are the co-dominant type and RAPD, AFLP are usually
dominant markers.
Comparison of molecular markers with biochemical
markers
Biochemical markers or isozyme markers were used for
the study of genetic variability or diversity, establishing
phylogenetic differences, population genetics, taxonomy,
development biology and for characterization of plant
genetic resources and plant breeding technology (Staub et
al., 1999). Isozymes are a structurally different form of an
enzyme with same catalytic functions and usually
originates from changes of amino acids causing
differences in net charge or conformation of the enzyme or
causing a change in their electrophoretic mobility
(Kennedy and Thompson, 1991). Enzymes are proteins
consisting of amino acids having net charge depending on

Hossain et al. 620
Table 1: Comparison of the five most widely used molecular marker in plants
Serial no RAPD AFLP RFLP MicrosatelliteSNP
Genomic abundance Very high Very high Very high Medium Medium
Amount of DNA required per reaction Very low (0.2µg) Medium (0.5-1.0 µg) High (10 µg) Low (0.5 µg) Low (0.5 µg)
Reproducibility Unreliable High High High Medium
Development cost Low Medium Low High High
Ease of use Labour intensive Initially difficult Easy Easy Easy
Amenable to automation Moderate Moderate Low High High
Cost per assay Low Moderate High Low Low
Polymorphism High Very high Medium High Medium
PCR based Yes Yes No Yes Yes
Inheritance Dominant Co-dominant Codominant Dominant Dominant
Cloning or Sequencing No No Yes Yes No
Utility for genetic mapping Cross specific Cross specific Species
specific
Species
specific
Cross
specific
Detection of alleles No No Yes Yes No
Radioactive detection No Yes/no Yes No No
the stretch of amino acids. When a mutation in the DNA
occurs, it changes the amino acid comprising the protein
thus changes the net charge of the amino acid that can be
detected by polyacrylamides gel electrophoresis. Because
of the change in the electric charge, the migration rate of
amino acid varies and different allelic variants can be
detected which are called iso-loci. However, allozyme
marker is sometimes referred to as isozyme variation (Li,
1999). Because of their consistency in the gene
expression and less responsive to the environmental
factors, allozyme markers were used extensively in the
past for plant breeding and genetic diversity study. The
main advantage of a biochemical marker is it simplicity.
DNA extraction and genome sequencing are not
mandatory for analysis of biochemical marker. The assay
is simple and quick. Allozyme markers are a co-dominant
marker and highly reproducible. Some of the allozyme
markers are inexpensive depending on the enzyme-
staining reagent used. Biochemical markers are the oldest
of all the molecular marker-based analysis and are
successfully applied in several crop improvement
programme (Glaszmann et al., 1989). However, low level
of polymorphism and low genomic abundance are the
limitation of biochemical and allozyme markers.
Biochemical markers are not genetic material and are
products of gene expression, so they are susceptible to
environmental factors (Naqvi, 2007).
Application of molecular markers in crop
improvement
Recent advancement of molecular biology has led to
development of molecular markers that have made plant
breeding more precise and faster. Among the most
promising markers, breeders use molecular genetic
markers to detect its presence or absence in plants of
interest of specific alleles and thus use them as selection
tools. Such a selection of favourable plants based on
linked markers is termed as Marker-Assisted Selection
(MAS). Genetic diversity studies are necessary to
determine the genetic distance among genotypes and to
identify species among related groups with similar genetic
backgrounds for conserving, determining and utilizing
germplasm for future breeding program. Molecular
markers techniques provide opportunities to obtain high
amplification of genetic traits (Tostain et al., 2003).
Development of high yielding varieties with desired
phenotypic characters and population is one of the major
aims in plant breeding technology of self as well as the
cross-pollinated crops (Waseem et al., 2012). Molecular
markers are used to select the plants with genomic regions
that are associated in the expression of traits of interest
(Choudhary et al., 2008). Later these selected individuals
are used to crosses in the breeding program. This is
achieved by pyramiding the desired gene interests. In
conventional breeding, it is based on the selection of elite
genotypes with segregating offspring obtained from the
cross. But this process is affected by genotype and
environmental factors that interfere with the result of the
experiment. In addition, this procedure is time-consuming,
often expensive for several phenotypic characters and
sometimes unreliable for some traits. Molecular marker is
not affected by the environmental factors and can be
detected in any stage of the plant. MAS greatly depends
upon the linkage relationship of the marker with the desired
traits. Potentiality of molecular marker depends upon the
capability of revealing polymorphism of the nucleotide
sequence for discrimination of different traits. Different
molecular markers are used widely for MAS. Five major
criteria are considered in selecting markers for MAS.
These are quantity and quality of DNA required, reliability,
cost, level of polymorphism and technical protocols for
marker assay (Zhang et al., 2017). Major markers used for
MAS includes RFLP, AFLP, RAPD, Microsatellite or SSR,
ISSR, SNP, CAPS) and other type of molecular marker.
MAS has much application in plant breeding such as in the
assessment of genetic diversity and parental selection,
variety identification, useful for the development of disease
and insect resistant crop plants, important for transferring
the desirable gene from wild-type into cultivar. MAS is also
very effective in transferring resistance to biotic and abiotic
stress of plants.

CONCLUSION
The accurate genomic identification of different plants is
an important task for their conservation and utilization.
Hitherto different methods are identified and developed at
DNA sequencing level. These techniques have introduced
many molecular marker systems for identification and
evaluating diversity relationship among different plant
species. During the past 15 years, microsatellite markers
are considered as the most suitable marker system among
all the molecular markers. This marker has high
polymorphism and co-dominant character, and are useful
for detection of homozygote and heterozygote. SNP
markers seem to very interesting and exciting marker but
the cost is not reasonable for developing countries. SCAR
markers can be used for the development and genetically
improvement of economically important crop plants. This
type of marker is usually cost-effective and also labour
intensive. So easily available and comparatively less
expensive marker systems like RAPD, SSR, ISSR, RFLP
marker become the choice of interest for scientist all over
the world. Also, the use of EST and EST based markers
like EST-SSR, EST-CAPS and EST-RFLP are applicable
only for some economically important crop plants which
genome sequence are available. With the increasing
development in genome sequencing and accessibility to
the GenBank, new molecular markers for specific genes or
coding regions of the genome are being designed. During
the last few decades, there has been a great appearance
in the advanced molecular methodology and this
development has greatly influenced research in the
different field of biological science like taxonomy, genetics,
ecology, plant breeding, bioinformatics, and other relevant
fields. Sequencing data obtained from the advanced
genome research will be useful for development of new
molecular marker systems which will be helpful for MAS
breeding for crop improvement and sustainable agriculture
in future.
DISCLOSURE STATEMENT
No potential conflict of interest was reported by the
authors.
ACKNOWLEDGEMENT
Authors gratefully acknowledge the research grant (No#
LS201628) for this study obtained from the Grant for
Advanced Research in Education, Ministry of Education,
Government of Bangladesh.
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Accepted 17 December 2019
Citation: Hossain MA, Hossen MS, Karim MR (2020).
Molecular Markers: Indispensable Tools for Genetic
Diversity Analysis and Crop Improvement Biotechnology.
International Journal of Plant Breeding and Crop Science,
7(1): 613-623.
Copyright: © 2020: Hossain et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.

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