What is Marker?

Marker is a piece of DNA

molecule that is
associated with a certain
trait of a organism
Morphological

Types of
Biochemical Markers
Genetic
 Definition of molecular marker

A sequence of DNA or protein that can be screened

to reveal key attributes of its state or composition
and thus used to reveal genetic variation.

 A molecular markers a DNA sequence that is

readily detected and whose inheritance can be
easily be monitored. The uses of molecular markers
are based on the naturally occurring DNA
polymorphism, which forms basis for designing
strategies to exploit for applied purposes.
 IDEAL MOLECULAR MARKER:
An ideal molecular marker must have some desirable properties.
1) Highly polymorphic nature:
A marker must to be polymorphic i.e. it must exit in different forms so
that chromosome carrying the mutant genes can be distinguished from
the chromosomes with the normal gene.
2) Co-dominant inheritance: determination of homozygous and
heterozygous states of diploid organisms.
3) Frequent occurrence in genome: A marker should be evenly and
frequently distributed throughout the genome.
4) Selective neutral behaviors: The DNA sequences of any organism are
neutral to environmental conditions or management practices.
5) Easy access (availability).
6) Easy and fast assay.
7) High reproducibility.
8) Easy exchange of data between laboratories.
 TYPES OF MOLECULAR MARKERS
• Due to rapid developments in the field of molecular
genetics, a variety of molecular markers has emerged
during the last few decades

Biochemical
marker
Allozyme
Traditional
marker
Non-PCR systems
based marker
RFLP, Minisatellite (VNTR)

PCR based
marker PCR
Microsatellite, RAPD, AFLP, CAPS, generation: in
ISSR, SSCP, SCAR, SNP, etc. vitro DNA
amplification
 RFLP (Non-PCR based marker)

• Targets variation in DNA restriction sites and in DNA

restriction fragments. Sequence variation affecting the
occurrence (absence or presence) of endonuclease
recognition sites is considered to be main cause of length
polymorphisms.

Techniques: Electrophoresis and

hybridization
• The technique centers around the digestion of genomic DNA digested with
restriction enzymes.

• These enzymes are isolated from bacteria and consistently cut DNA at specific
base pair sequences which are called recognition sites.

• These recognition sites are not associated with any type of gene and are
distributed randomly throughout the genome.

• When genomic DNA is digested with one of these restriction enzymes, a series
of fragment are produced of varying length.
• These fragments are separated using agarose or polyacrylamide gel
electrophoresis (PAGE) and yield a characteristic pattern.
• Variations in the characteristic pattern of a RFLP digest can be caused by
 base pair deletions,
 mutations,
 translocations and transpositions
which result in the loss or gain of a recognition site resulting in a fragment of
different length and polymorphism.
 Advantages:
Moderately polymorphic
High genomic abundance and random distribution
 Co-dominant
 High reproducibility.
 Reliable markers in linkage analysis and breeding.

Disadvantages:
large quantities (1–10 μg) of purified, high molecular weight DNA are required for
each DNA digestion and Southern blotting.
Larger quantities are needed for species with larger genomes and for the greater
number of times needed to probe each blot.
The requirement of radioactive isotope makes the analysis relatively expensive and
hazardous.
 The assay is time-consuming and labour-intensive and only one out of several
markers may be polymorphic, which is highly inconvenient especially for crosses
between closely related species.
Their inability to detect single base changes restricts their use in detecting point
mutations occurring within the regions at which they are detecting polymorphism.
 Applications:

RFLPs can be applied in diversity and phylogenetic studies ranging from

individuals within populations or species, to closely related species.

RFLPs have been widely used in gene mapping studies because of their high
genomic abundance due to the ample availability of different restriction enzymes and
random distribution throughout the genome (Neale & Williams 1991).

 They also have been used to investigate relationships of closely related taxa
(Miller & Tanksley 1990; Lanner et al. 1997), as fingerprinting tools (Fang et al.
1997), for diversity studies (Debreuil et al. 1996), and for studies of hybridization,
including studies of gene flow between crops and weeds (Brubaker & Wendel 1994,
Clausen & Spooner 1998, Desplanque et al. 1999).
 RAPD (PCR-based marker)
Uses primers of random sequence to amplify DNA
fragments by PCR. Polymorphisms are considered to be
primarily due to variation in the primer annealing sites, but
they can also be generated by length differences in the
amplified sequence between primer annealing sites

Techniques: PCR and

Electrophoresis
• RAPD was the first PCR based molecular marker technique
developed and it is by far the simplest.

• Short PCR primers (approximately 10 bases) are randomly and

arbitrarily selected to amplify random DNA segments throughout
the genome.

• The resulting amplification product is generated at the region

flanking a part of the 10 bp priming sites in the appropriate
orientation.

• RAPD often shows a dominant relationship due to primer being

unable to bind on recessive alleles.

• RAPD products are usually visualized on agarose gels stained with

ethidium bromide.
 Advantages:

Quick and easy to assay.

Low quantities of template DNA are required, usually 5–50 ng per
reaction.
Since random primers are commercially available, no sequence data for
primer construction are needed.
RAPDs have a very high genomic abundance and are randomly
distributed throughout the genome.
Dominant markers
 Disadvantages:
 low reproducibility
highly standardized experimental procedures are needed because of their
sensitivity to the reaction conditions.
require purified, high molecular weight DNA, and precautions are needed
to avoid contamination of DNA samples because short random primers are
used that are able to amplify DNA fragments in a variety of organisms.
the inherent problems of reproducibility make RAPDs unsuitable markers
for transference or comparison of results among research teams working in a
similar species and subject.
RAPD markers are not locus-specific, band profiles cannot be interpreted
in terms of loci and alleles (dominance of markers).
 Applications:

The application of RAPDs and their related modified markers in

variability analysis and individual-specific genotyping has largely been
carried out, but is less popular due to problems such as poor
reproducibility, faint or fuzzy products, and difficulty in scoring bands,
which lead to inappropriate inferences.
RAPDs have been used for many purposes, ranging from studies at the
individual level (e.g. genetic identity) to studies involving closely related
species.
RAPDs have also been applied in gene mapping studies to fill gaps not
covered by other markers (Williams et al. 1990, Hadrys et al. 1992).

Variants of the RAPD technique include Arbitrarily Primed Polymerase

Chain Reaction (AP-PCR), which uses longer arbitrary primers than
RAPDs, and DNA Amplification Fingerprinting (DAF) that uses shorter,
5–8 bp primers to generate a larger number of fragments. Multiple
Arbitrary Amplicon Profiling (MAAP) is the collective term for
techniques using single arbitrary primers.
 Microsatellites or Simple sequence Repeat (SSR)

The term microsatellites was coined by Litt & Lutty (1989) and it also
known as Simple Sequence Repeats (SSRs), are sections of DNA,
consisting of tandemly repeating mono-, di-, tri-, tetra- or penta-nucleotide
units that are arranged throughout the genomes of most eukaryotic species.
 Microsatellite polymorphism can be detected by Southern hybridisation
or PCR.
Microsatellites, like minisatellites, represent tandem repeats, but their
repeat motifs are shorter (1–6 base pairs). If nucleotide sequences in the
flanking regions of the microsatellite are known, specific primers (generally
20–25 bp) can be designed to amplify the microsatellite by PCR.
 Advantages:

codominance of alleles, their high genomic abundance in eukaryotes and

their random distribution throughout the genome.
Due to the use of long PCR primers, the reproducibility of microsatellites is
high and analysis do not require high quality DNA.
The screening of microsatellite variation can be automatedby using
automatic sequencers like EST-SSR markers are one class of marker that can
contribute to ‘direct allele selection’.
Yu et al. (2004) identified two EST-SSR markers linked to the photoperiod
response gene (ppd) in wheat.
 Disadvantages:
One of the main drawbacks of microsatellites is that high
development costs are involved if adequate primer sequences for the
species of interest are unavailable, making them difficult to apply to
unstudied groups.
Although microsatellites are in principle co-dominant markers,
mutations in the primer annealing sites may result in the occurrence
of null alleles (no amplification of the intended PCR product), which
may lead to errors in genotype scoring.
A very common observation in microsatellite analysis is the
appearance of stutter bands that are artifacts in the technique that
occur by DNA slippage during PCR amplification. These can
complicate the interpretation of the band profiles because size
determination of the fragments is more difficult and heterozygotes
may be confused with homozygotes.
 Application
•In general, microsatellites show a high level of polymorphism. As a
consequence, they are very informative markers that can be used for many
population genetics studies, ranging from the individual level to that of closely
related species.
•Microsatellites are also considered ideal markers in gene mapping studies
(Hearne et al. 1992, Morgante & Olivieri 1993, Jarne & Lagoda 1996).
•Molecular markers have proven useful for assessment of genetic variation in
germplasm collections (Mohammadi & Prasanna 2003).
• Expansion and contraction of SSR repeats in genes of known function can be
tested for association with phenotypic variation or, more desirably, biological
function (Ayers et al.1997).
•Several studies have found that SSRs are useful for estimating genetic
relationship and at the same time provide opportunities to examine functional
diversity in relation to adaptive variation (Eujayl et al.2001, Russell et al.
2004).