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What are Molecular Markers?
Specific fragments of DNA that can be identified within the whole
Molecular markers are the general assays that allow detection of the
sequence differences between two or more individual.
Molecular markers are found at specific locations of the genome.
They are used to 'flag' the position of a particular gene or the
inheritance of a particular characteristic or desired characteristics
A molecular marker is a DNA sequence that is readily detected and
whose inheritance can easily be monitored .
Marker systems are tools which is used to mark a trait in living organism
Variation in macro-molecules
BIOCHEMICAL MARKERS GENETIC MARKERS
They are protein produced by expression of gene
Depend upon sequence of DNA
•Requires expression of trait / gene
•Expression sex limited
•Expressed late in life
RFLP- Restriction fragment length polymorphism.
RAPD- Random amplification of polymorphic DNA.
AFLP-Amplified fragment length polymorphism.
SCAR-Sequence characterize amplified region.
STS- Sequence tagged sites.
EST-Express sequence tags.
SNP-Single nucleotide polymorphism.
SSR-Simple sequence repeats
CAPS-Cleaved amplified polymorphic sequences.
it must be polymorphic.
A marker should be evenly and frequently
distributed throughout the genome.
It should be easy, fast and cheap to
It should be reproducible.
High exchange of data between
Properties of Ideal Genetic Marker
Feature RFLP RAPD AFLP SSR or
DNA required (µg) 10 0.02 0.5-1.0 0.05
DNA quality High High Moderate Moderate
PCR-based No Yes Yes Yes
No. of polymorphic loci
1.0-3.0 1.5-50 20-100 1.0-3.0
Ease of use Not easy Easy Easy Easy
Reproductibily High Unreliable High High
Development cost Low Low Moderate High
Cost per analysis High Low Moderate Low
Table 1 : Comparision of the most broadly used techniques
of molecular markers
The variation(s) in the length of DNA fragments produced by a specific
restriction endonuclease from genomic DNA s of two or more
individuals of a species
Restriction fragment length polymorphism (RFLP) technology was first developed in
the 1980s for use in human genetic applications and was later applied to plants.
By digesting total DNA with specific restriction enzymes, an unlimited number of
RFLPs can be generated.
RFLPs are relatively small in size and are co-dominant in nature.
If two individuals differ by as little as a single nucleotide in the restriction site, the
restriction enzyme will cut the DNA of one but not the other. Restriction fragments of
different lengths are thus generated.
All RFLP markers are analyzed using a common technique. However, the analysis
requires a relatively complex technique that is time consuming and expensive.
The hybridization results can be visualized by
1. Autoradiography (if the probes are radioactively labeled), or
2. Chemiluminesence (if non-radioactive, enzyme-link methods are used for
probe labeling and detection).
Any of the visualization techniques will give the same results. The visualization
techniques used will depend on the laboratory condition
-simple method as no sequence specific information is required
-it is not depend on PCR
-it required large amount of highly pure DNA
-it require constant supply of probes
-it is laborious to identify suitable markers
-it is time consuming
-it requires expertise in autoradiography
Applications of molecular markers
►MEASURE OF GENETIC DIVERSITY
►GENOTYPIC PYRAMIDYING AND
►INDIRECT SELECTION USING QUANTITATIVE
TRAITS LOCI (QTLS)
►IDENTIFICATION OF GENOTYPE
Any DNA segment that is amplified using short oligodeoxynucleotide primer
of arbitrary nucleotide sequence (amplifier) and polymerase chain reaction
procedure (Khal, 2001)
RAPD also known as,
AP-PCR(Arbitrarily primed PCR),
DAF (DNA amplification fingerprinting)&
MAAP(Multiple arbitrary amplicon profiling)
RAPD are a dominant marker system
1. RAPDs are produce by PCR using genomic DNA and arbitrary primers
2. Taq polymerase is used to amplify DNA segment between closely spaced
sequence (< 2kb) and complementary to the short random oligomers (typically
3. RAPD polymorphism result from change in the primer-binding site in the DNA
In variety A there are 4 primer binding sites resulting in two RAPD products, variety
B lacks one of the binding sites resulting in only one RAPD marker being produced
Primers point in the same
direction, so amplification
Primers too far apart, so
amplification won’t happen
> 2,000 bases
Primers are just the
right distance apart,
so fragment is
100 - 1,500 bases
1) Master Stock Mixture
2) Add 25µl of master mix to 5µl of your DNA in a sterile tube
Note: In each PCR run you conduct, include 2 sample, one of control DNA
without primer (3µl DNA), and one sample without DNA (5µl ddH2O)
-need small amount of DNA
-it involves non-radioactive assay
-it does not required specific probe libraries
-it provide quick and efficient screening for DNA
sequence based on polymorphism at many loci
-it is inherited as dominant traits
-there is a bands due to relatively short primer
-the production of non-parental bands in the offspring of
known pedigree warrants its use with extreme care
-it is sensitive to change in PCR conditions
Any difference between corresponding DNA fragment from two
organisms A & B that is detected by amplified restriction length
1. The amplified fragment length polymorphism technique combines components
of RFLP analysis with PCR technology.
2. Total genomic DNA is digested with a pair of restriction enzymes normally a
frequent and rare cutter.
3. Adaptors of known sequence are then ligation to the DNA fragments.
4. Primer complementary to the adaptors are used to amplify the restriction
5. The PCR amplified fragments can then separated by gel electrophoresis and
banding patterns visualized.
6. A range of enzymes and primer are available to manipulate the complexity of
AFLP fingerprint to suit application
For restriction digestion we use two type of cutter i.e
•Rare cutter (6bp) EcoRI
•Frequent cutter (4bp) MseI
Interstitial cohesive ends
PCR amplification using EcoRI/MseI
-it has a wide scale applicability
-it discriminates heterozygotes from
homozygotes when a gel scanner is used
-used for mapping
-it is highly expensive
-it required more DNA than RAPD
-it required experience of sequencing gels
Microsatellites can be amplified for identification by the polymerase
chain reaction (PCR) process, using the unique sequences of flanking
regions as primers
DNA is repeatedly denatured at a high temperature to separate the
double strand, then cooled to allow annealing of primers and the
extension of nucleotide sequences through the microsatellite.
This process results in production of enough DNA to be visible
on agarose or polyacrylamide gels.
With the abundance of PCR technology, primers that flank
microsatellite loci are simple and quick to use, but the development of
correctly functioning primers is often a tedious and costly process.
-simple and easy to use
-easy to detect via PCR
-perfectly suited for used in map-based cloning
-cost is higher for establishing polymorphic primer sites
and investment in the synthesizing the oligonucleotides
-initial identification, DNA sequence information
Application of SSR
Assessment of genetic variability and characterization of germplasm.
Identification and fingerprinting of genotypes.
Estimation of genetic distances between population, inbreeds and
Marker assisted selection.
Identification of sequence of useful candidate genes
An autoradiograph detecting parent (P1&P2)
and homozygous and heterozygous (H ) F1
M R H H S R H H S R H H S H R R H R H
Fig: Identification of RAPD marker link to brown plant hoper resistance
gene in rice
June et al.,2003
RAPD-ANALYSIS OF GENETIC VARIATION OF FOUR IMPORTANT RICE
VARIETIES USING RAPD PRIMERS
Amplified RAPD patterns of OPR1
M - 1 Kb DNA Ladder
1 - ADT38
2 - ASD16
3 - IR20
4 - PONNI
Amplified RAPD patterns of OPR2.
Mani et al. (2010)Tamil Nadu
UPGMA dandogram based on Nei’s
(1978) original measure of genetic
distance, summarizing the
data on differentiation between four
samples of O. sativa genotypes according
to RAPD analysis.
Genetic distance between O. sativa populations of four different
rice varieties based on Nei’s 1978 measures of genetic distance.
PONNI IR-20 ADT38 ASD16
PONNI 0.3913 1.7776 1.02564
IR-20 0.3913 1.60944 1.95601
ADT38 1.77767 1.60944 0.8574
ASD16 1.02564 1.95601 0.8574
1 2 3 4 5
Fig: Molecular mapping of fertility restorer gene in basmati rice
using micro satellite marker.
Delhi Mishra et al.2001
A Rice microsatellite marker RM 258 identified to be linked with fertility
restorer gene in PRR- 78 using bulk segregant analysis.
DNA marker (lane 1). Restorer line PRR 78 (lane 2).
CMS line IR 58025 (lane 3).
Fertile bulk showing heterozygous pattern (lane 4).
Sterile bulk showing homozygous pattern (lane 5)
Fig. 7 : Hybrids identification by using seed protein markers
Bhubaneshwar Panigrahi et al., 2001
Easy to detect via PCR
Relatively expensive and time consuming to develop
Initial identification, DNA sequence information necessary
PCR: Polymerase Chain Reaction
“Amplify” DNA by in-vitro (in plastico) synthesis
enzyme: Taq DNA polymerase, not denatured at high
temps used to denature DNA
primers: short (~ 20 b) oligonucleotides bind to
denatured DNA, required to start DNA synthesis