Int.J.Curr.Microbiol.App.

Sci (2018) Special Issue-6: 816-825

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Special Issue-6 pp. 816-825
Journal homepage: http://www.ijcmas.com

Review Article
Molecular Marker Techniques: A Review

R. R. Dhutmal*, A. G. Mundhe and A. W. More

Sorghum Research Station, Vasantrao Naik Marathwada Krishi Vidyapeeth,

Parbhani-431401 (M.S.), India
*Corresponding author

ABSTRACT

Several marker techniques have been generated in the last decade starting from the first
generation molecular marker, RFLP, that was based on DNA-DNA hybridization and later
the invention of PCR gave rise to a second generation of faster and less expensive PCR-
based markers followed by the third generation sequence based makers. Following the rise
of EST databases and whole genome sequencing projects several functional markers have
been developed. However, marker techniques are continuously changing and evolving.
Among the various marker techniques that are available, particularly promising are
Keywords Amplified Fragment Length Polymorphisms, Random Amplified Polymorphic DNA,
Molecular Microsatellites, Sequence Characterized Amplified Region or Sequence Tagged Sites etc.
maker The most appropriate genetic marker technique to be used will depend on the specific
technique, application, the presumed level of polymorphism required, the presence of sufficient
REPL, PCR, technical facilities and know-how, time constraints and financial limitations. Molecular
DNA markers have become important tools for a large number of applications ranging
phylogenetic analysis, diversity studies, construction of genetic maps, comparative maps,
framework maps, framework/region specific mapping, novel allele detections, high-
resolution mapping, very fast mapping, region-specific marker saturation, gene tagging,
marker-assisted selection, map-based gene cloning, varietal/line identification, hybrid
identification, seed testing, fingerprinting, alien gene introduction etc. Thus molecular
markers are considered as valuable tools for genome analysis even in crops where the
whole genomes are sequenced.

Introduction

The era of genomics started with the morphological and molecular.

development of genetic tools like the DNA- Morphological markers are those traits
based molecular markers that have been which are visible to the naked eyes while
extensively used in various fields like molecular markers are those which
taxonomy physiology, embryology, genetic expresses at the molecular level (i.e. DNA
engineering, etc. Markers are the traits or proteins). A molecular marker is defined
which can be used to distinguish or as a particular segment of DNA that is
differentiate between the populations under representative of the differences at the
study. Markers are the traits which can be genome analysis. The discovery of PCR
used to distinguish or differentiate between (polymerase chain reaction) was a landmark
the populations under study. Markers are in this effort and proved to be a unique
broadly divided into two major category i.e. process that brought about a new class of

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DNA profiling markers. The random DNA of molecular markers from the genes that are
based markers were the first genomic tools responsible for the expression of phenotypic
that were developed and used for several trait variations. These markers from
purposes in crop plants. Molecular markers transcribed region of the genome, target the
offer numerous advantages over functional polymorphism in the gene
conventional phenotype based alternatives sequences and allows selection in different
as they are stable and detectable in all genetic backgrounds which isnot always
tissues regardless of growth, differentiation, possible random markers.
development, or defense status of the cell.
Properties of molecular markers
They are not confounded by the
environment, pleiotropic and epistatic Properties of ideal genetic markers
effects. Markers are ideal tools for
examining the relationships among There are certain unique properties which
individuals, populations or phylogenetic makes any marker as ideal genetic marker. It
taxa. An increasing number of monogenic, should not have any detrimental effect on
race-specific genes showing gene-for-gene phenotype, co-dominant in expression,
interaction have been mapped and single copy, economic to use, highly
agronomically important genes have been polymorphic, easily assayed, multi-
correlated using molecular markers. These functional, highly available (Un-restricted
are widely being used in screening programs use). Genome-specific in nature (especially
for selecting disease resistant clones and when working with polyploids), can be
introgression of several resistant genes into a multiplexed and its ability to be automated.
particular background.
Genomic abundance
Most of the above mentioned markers are
developed from genomic DNA, and The number of markers that can be
therefore they may arise from both the generated is determined mainly by the
transcribed regions and the non-transcribed frequency at which the sites of interest occur
regions of the genome. These DNA-based within the genome. RFLPs and AFLPs
markers derived from any region of the generate abundant markers due to the large
genome have also been described as RDMS. number of restriction enzymes available and
These markers when used for indirect the frequent occurrence of their recognition
selection are completely independent of any sites within genomes. Within eukaryotic
functional knowledge about the underlying genomes, microsatellites have also been
DNA sequences. found to occur frequently. RAPD markers
are another abundant class of markers
Thus even for tightly linked markers, the because numerous random sequences can be
effectiveness of marker aided selection is used for primer construction. If, in addition
greatly diminished by the occasional to genomic abundance, genome coverage is
uncoupling of the marker form the trait also sought, caution should be taken in
during many cycles of meiosis in the marker selection. While some markers are
breeding program. Also the application of known to be scattered quite evenly across
such random markers for selection across the genomes, others, such as some AFLP
populations has been limited. Recently, markers, sometimes cluster in certain
interest has shifted towards the development genomic regions.

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Level of polymorphism species as well as from random

amplification of the genome. These
The resolving power of genetic markers is techniques include the first generation
determined by the level of polymorphism restriction based markers like RFLP
detected which is in turn determined by the followed by the second generation
mutation rate at the genomic sties involved. amplification based markers like RAPD,
Mutation at mini satellite and microsatellite AFLP. SSR, ISSR and the third generation
loci, occur due to changes in the number of sequence based markers like SNPs. The
repeat units of the core sequence, and have RFLP markers which are highly
been estimated to occur at the relatively high polymorphic, reproducible and are co-
frequency. Higher resolving power is dominantly inherited but have been used less
required when samples are more closely frequently owing to the laborious procedure
related. For example, analyses within involved. RAPDs and ISSRs had been the
species or among closely related species marker of choice in the nineties and soon
may call for fast evolving markers such as interest had shifted to more reliable and
microsatellites. However if the objective is reproducible marker systems like AFLPs
to study genetic relatedness at higher and SSRs.
taxonomic levels, AFLPs or RFLPs may be
a better choice because co-migrating fast- Hybridization based markers
evolving markers will have less chance of
being homologous. A primary guiding In hybridization based markers, DNA
principle in marker selection is that more profiles are visualized by hybridizing the
conservative markers (those having slower restriction enzymes digested DNA to a
evolutionary rates) are needed with labeled probe, which can be DNA fragment
increasing evolutionary distance and vice- of known origin or sequence. Restriction
versa. fragment length polymorphism (RFLP) was
the first hybridization based marker system.
Codominance of Alleles Later on many markers probes such as
microsatellites, mini satellites and STS were
Codominance markers are markers for developed initially as hybridization based
which both alleles are expressed when co- marker. Within the development of easier
occurring in an individual. Therefore, with cloning and sequencing techniques and
codominant markers, heterozygotes can be availability of sequence databases these
distinguished from homozygotes, allowing markers have been converted in to PCR
the determination of genotypes and allele based markers which are easier to assay.
frequencies at loci. Codominant markers are
preferred for most applications. The Restriction Fragment Length
majority of codominant markers are single Polymorphism (RFLP)
locus markers and hence the degree of
information per assay is usually lower The publication of Botstein et al., (1980)
compared to the multilocus techniques. about the construction of genetic maps using
restriction fragment length polymorphism
Molecular markers as genomic tools was the first reported molecular marker
technique in the detection of DNA
Several molecular marker techniques were polymorphism. In RFLP, DNA
developed from the genome of the crop polymorphism is detected by hybridizing a

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chemically labeled DNA probe to a closely related taxa, as fingerprinting tools,

Southern blot of DNA digested by for diversity studies, and for studies, and for
restriction endonucleases, resulting in studies of hybridization and introgression,
differential DNA fragment profile. Labelling including studies of gene flow between
of the probe may be performed with a crops and weeds.
radioactive isotope or with alternative non-
radioactive stains, such as digoxigenin or Amplification based markers
fluorescein. Probes are generated through
the construction of genomic of The in vitro amplification of the DNA by
complementary DNA (cDNA) libraries and polymerase chain reaction (PCR) has proven
hence may be composed of specific to be revolutionary technique in molecular
sequence of unknown identity (genomic biology. The amplified products can be
DNA) or part of the sequence of a functional visualized on a gel in the form of bands.
gene (exons only, cDNA). The hybridization PCR based markers involve in vitro
results can be visualized by autoradiography amplification of particular DNA sequences
(if the probes are radioactively labelled), or or loci with the help of specially or
using chemiluminesence (if nonradioactive, arbitrarily chosen oligonucleotide primers
enzyme-linked methods are used for probe and a thermo stable DNA polymerase
labeling and detection). RFLPs correspond enzyme. The amplified products are
to DNA fragments, usually within the range separated electrophoretically and banding
of 2-10 kb, that have resulted from the patterns are detected by different methods
digestion of genomic DNA with restriction such as staining and autoradiography. The
enzymes. The differential profile is PCR based markers can be divided in two
generated due to nucleotide substitutions or categories viz., sequence arbitrary markers
DNA rearrangements like insertion or and sequence dependent markers.
deletion or single nucleotide
polymorphisms. The RFLPs markers are Random Amplified Polymorphic DNA
relatively highly polymorphic, (RAPD)
codominanltly inherited and highly
reproducible. RFLPs are applied in diversity The RAPD technique is based on PCR
and phylogenetic studies ranging from amplification of genomic DNA. It deduces
individuals within populations or species, to DNA polymorphisms produced by
closely related species. RFLPs have been "rearrangements or deletions at or between
widely used in gene mapping studies oligonucleotide primer binding sites in the
because of their high genomic abundance genome” using short random
due to the ample availability of different oligonucleotide sequences (mostly ten bases
restriction enzymes and random distribution long) (Williams et al., 1990). As the
throughout the genome (Neale and Williams approach requires no prior knowledge of the
1991). Botstein et al., (1980) used RFLP genome that is being analyzed, it can be
markers for the first time in the construction employed across species using universal
of genetic map. These are very reliable primers. The major drawback of the method
marker in linkages analysis and can is that the profiling is dependent on the
determine if linked gene is present in reaction conditions so may vary within two
homozygous or heterozygous stage due to different laboratories. Nevertheless due to
their codominant behavior. They also have the speed and efficiency of RAPD analysis,
been used to investigate relationship of high-density genetic maps has been

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developed in many plant species. Variants of motifs followed by a two-step PCR

the RAPD technique include Arbitrarily amplification of selected fragments. The
Primed Polymerase Chain Reaction (AP- selective amplification uses primers
PCR) which uses longer arbitrary primers composed of the adapters and 1 to 3 selected
than RAPDs, and DNA Amplification nucleotides at the 3’ end. It limits the
Fingerprinting (DAF) that uses shorter, 5-8 number of fragments to a resolvable range.
bp primers to generate a larger number of The PCR-amplified fragments can then be
fragments. Randomly amplified separated by gel electrophoresis and banding
microsatellite polymorphisms (RAMPO) patterns visualized. A range of enzymes and
another variant of RAPD and is a PCR- primers are available to manipulate the
based strategy where the genomic DNA is complexity of AFLP fingerprints to suit
first amplified using arbitrary (RAPD) application. The AFLP banding profiles are
primers and the amplified products are then the result of variations in the restriction sites
electrophoretically separated and the dried or in the intervening region.
gel is hybridized with microsatellite
oligonucleotide probes. The AFLP technique simultaneously
generates fragments from many genomic
Several advantages of oligonucleotide sites (usually 50-100 fragments per reaction)
fingerprinting, RAPD and microsatellite that are separated by polyacrylamide gel
primed PCR are combined, making it electrophoresis and that are generally scored
speedy, highly sensitive, detecting higher as dominant markers. However, by use of
variability and requires no prior DNA automatic gel scanner heterozygote may be
sequence information. This technique has distinguished from homozygote based on
been successfully employed in the genetic band intensity differences, which facilitates
fingerprinting of closely related genotypes the scoring of many AFLPs as codominant
of several crop species. Multiple Arbitrary markers. The AFLP technique generates
Amplicon Profiling (MAAP) is the fingerprints of any DNA regardless of its
collective term for techniques using single source, and without any prior knowledge of
arbitrary primers. DNA sequence. Most AFLP fragments
correspond to unique positions on the
Amplified Fragment Length genome and hence can be exploited as
Polymorphisms (AFLP) landmarks in genetic and physical mapping.

To overcome the limitation of Selectively Amplified Microsatellite

reproducibility associated with RAPD, Polymorphic Locus (SAMPL)
AFLP technology (Vos et al., 1995) was
developed. It combines the power of RFLP This technique is a variant of AFLP
with the flexibility of PCR-based technology technique which amplifies microsatellite loci
by ligating primer recognition sequences by using a single AFLP primer in
(adaptors) to the restricted DNA and combination with a primer complementary
selective PCR amplification of restriction to compound microsatellite sequences,
fragments using a limited set of primers. The which do not require prior cloning and
DNA is cut with two restriction enzymes, characterization. It is considered more
one being a frequent cutter and the other an applicable to intraspecific than to
infrequent cutter. This is followed by interspecific studies due to frequent null
ligation of adapters, including restriction alleles.

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Single-Strand Conformation Polymorphism absence of fragments of particular size. PCR

(SSCP) products may be separated on agarose gel
with ethidium bromide visualization, or
SSCP relies on intra strand (single strand) polyacrylamide gel with silver staining
differences (conformation) in DNA of techniques. The ISSR marker technique is
different sequence. SSCPs are DNA nearly identical to RAPD expect that ISSR
fragments of about 200-800 bp amplified by primer sequences are designed from
PCR using specific primers of 20-25 bp. Gel microsatellite regions and the annealing
electrophoresis of single-strand DNA is used temperatures used are higher which makes
to detect nucleotide sequence variation them more robust. The advantage of greater
among the amplified fragments. The band amplification is augmented.
electrophoretic mobility of single-strand Techniques related to ISSR analysis are
DNA depends on the secondary structure Single Primer Amplification Reaction
(conformation) of the molecule, which is (SPAR) that uses a single primer containing
changed significantly with mutation. Thus, it only the core motif of a microsatellite, and
provides a method to detect nucleotide Directed Amplification of Mini satellite
variation among DNA samples without region DNA (DAMD) that uses a single
having to perform sequence reactions. In primer containing only the core motif of a
SSCP the amplified DNA is first denatured, mini satellite. The main advantage of ISSRs
and then subject to non-denaturing gel is that no sequence data for primer
electrophoresis. construction are needed and requires low
quantities of DNA.
Inter Simple Sequence Repeats (ISSR)
Simple Sequence Repeat markers (SSR)
ISSRs are DNA fragments of about 100- or microsatellites
3000 bp located between adjacent,
oppositely oriented microsatellite regions. Microsatellites are short tandem repeats of
ISSRs are amplified by PCR using 1-6 base pairs. Di-, tri- or tetranucleotide
microsatellite core sequences as primers repeats like (CA)n, (AAT)n or (GATA)n are
with a few selective nucleotides as anchors widely distributed throughout the genome of
into the non-repeat adjacent regions (16-18 the plants and animals and developed as
bp). Primers based on repeat sequences, markers by deducing the flanking regions of
such as (CA) n, can be made with a the microsatellite. Their polymorphism lies
degenerate 3’-anchor, such as (CA) 8 RG or in the variation of the number of repeats,
(AGC) 6TY. An unlimited number of probably because of errors during
primers can be designed for various replication. If nucleotide sequences in the
combinations of di-, tri-, tetra and flanking regions of the microsatellite are
pentanucleotides with an anchor made up of known, specific primers (generally 20-25
few bases. The resultant PCR amplifies the bp) can be designed to amplify the
sequence between two SSR, yielding microsatellite by PCR. Microsatellite and
multilocus marker system useful for their flanking sequences can be identified by
fingerprinting, diversity analysis and constructing a small-insert genomic library,
genome mapping. About 10-60 fragments screening the library with a synthetically
from multiple loci are generated labelled oligonucleotide repeat and
simultaneously, separated by gel sequencing the positive clones.
electrophoresis and scored as the presence or Alternatively, microsatellites may be

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identified by screening sequence databases sequences as genetical markers involves the

for microsatellite sequence motifs from identification of polymorphic sequence that
which adjacent primers may then be affect the plant phenotype within these
designed. Polymerase slippage during DNA genes and validating the associations
replication, or slipped strand mispairing, is between these genes and trait variations.
considered to be the main cause of variation Recent studies show that these markers are
in the number of repeat units of a superior than the random DNA based
microsatellite resulting in length markers for marker assisted selections. The
polymorphisms. The advantages of SSRs raw sequence information will also aid in
include codominance of alleles, high screening for the occurrence of
genomic abundance in eukaryotes and microsatellite sequences (EST-SSR) or
random distribution throughout the genome, single nucleotide polymorphisms (EST-
with preferential association in low-copy SNP), after which markers can be developed
regions. It requires low quantities of that are targeted to transcribed regions of the
template DNA (10-100 ng per reaction) and genome.
the use of long PCR primers ensures the
reproducibility of microsatellites permitting Single Nucleotide Polymorphisms (SNPs)
exchange between researchers. SSR assays
are more robust than random amplified Single nucleotide polymorphisms are
polymorphic DNA (RAPD) and more variations in genome sequence of
transferable than AFLPs. Multiple individuals of a population. They constitute
microsatellites may be multiplexed during the most abundant molecular markers in the
PCR or gel electrophoresis if the size ranges genome and are widely distributed
of the alleles of different loci do not overlap throughout genomes although their
thus decreasing the analytical costs. occurrence and distribution varies among
Furthermore, the screening of microsatellite species. Maize has 1 SNP per 60-120 bp,
variation can be automated, if the use of while humans have an estimated 1 SNP per
automatic sequencers is an option. 1,000 bp. The SNPs are usually more
prevalent in the non-coding regions of the
Sequence based functional markers genome. Improvements in sequencing
technology and availability of an increasing
Expressed Sequence Tags (ESTs) number of sequences in the public domain
have made direct analysis of genetic
Expressed Sequence Tags are the result of variation at the DNA sequence level
sequencing cDNA clones and the possible. High throughput genotyping
information generated is generally stored in methods, including DNA chips, allele-
databases. The availability of sequence data specific PCR and primer extension
of expressed DNA has enabled the approaches make single nucleotide
development of markers that are physically polymorphisms (SNPs) especially attractive
associated with coding regions of the as genetic markers.
genome. These sequences can then be used
for designing primers to readily generate cDNA-AFLP
polymorphic markers. These markers are a
part of the cDNA/EST sequences and Complementary DNA can also be used as
represent functional component of the template for subsequent direct marker
genome. The development of these generation, for example through AFLP

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technology. cDNA-AFLP is commonly used Target Region Amplification

in the identification of genetic Polymorphism (TRAP)
polymorphisms between contrasting
phenotypes like resistant and susceptible Another related technique that uses EST
individuals under controlled conditions in sequence information is Target Region
order to facilitate the construction of linkage Amplification Polymorphism (TRAP). For
maps or to identify candidate resistant TRAP analysis, a fixed primer designed
genes. from a targeted EST sequence is combined
with an arbitrary primer having an AT- or
In sugarcane differentially expressed cDNA CG-rich core sequence. For different plant
fragments identified during biotic challenge species TRAP revealed multiple scorable
show great potential as genetic markers for fragments, and the technique may be well
pest and disease resistance. The sequences suited for determining the genotypes of
thus isolated have shown associations with germplasm and tagging genes for traits of
smut resistance and sugarcane mosaic virus interest. TRAP markers have been used for
resistance. tagging genes that confer resistance to
bacterial, fungal and viral pathogens in
Sequence-Related Amplified Polymorphism common bean.
(SRAP)
Resistance Gene Homologue
A simple PCR-based marker technique that Polymorphism (RGHP)
targets coding sequences in the genome is
Sequence-Related Amplified Polymorphism. The Resistance Gene Homologue
SRAP uses forward primers consisting of an Polymorphism is based on the availability of
unspecific filler sequence of ten bases, the candidate resistant gene sequences. The use
sequence CCGG and three selective of candidate genes as markers is more
nucleotides. effective in the case of disease resistance
since many genes involved in the resistant
Reverse primers also contain a filler pathways have been characterized. RGHPs
sequence, but are followed by the sequence target groups of resistance genes by PCR,
AATT and three selective nucleotides. The using primers for conserved domains of
CCGG sequence is used to target GC-rich resistance genes, such as the Leucine Rich
regions, such as exons in open reading Repeat (LRR) or the Nucleotide Binding
frames, while the AATT sequence on the Site (NBS), both involved in resistance
reverse primers is aimed at AT- rich regions, mechanisms. These RGHPs are then used to
such as promoters and introns. identify linkage with known disease
resistant loci for use in marker assisted
The generally conserved nature of exon selections as well as to clone the resistant
sequences, combined with the generally genes. Many RHGPs have been located to
variable nature of introns, promoters and chromosome regions containing major R
spacers, enables SRAP analysis to generate genes as well as QTLs. The cosegregation of
polymorphic bands. In Brassica oleracea L. RHGPs with major disease resistant genes
sequence analysis revealed that 45% of the and quantitative trait loci (QTL) has been
SRAP fragments could be matched with reported in several crops species (Pflieger et
known genes. al., 2001). The disease R-gene database
(available on line from the National Center

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for Genome Resources web site: for both pathogen recognition (resistance
http://www.ncgr.org/research/rgenes) genes and homologs) and plant defense
facilitates access to R-gene and R-gene-like responses (defense genes). These sequences
sequence data collected from public were mapped on the potato genome and
sequence and protein databases. The second their position was compared to those of
database contains information about genes resistance QTLs (Nelson et al., 1999).

Random DNA Markers (RDMs)

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