Dr.

Vaishali
Associate Professor, Biotechnology
SVPUA&T, Meerut
What is Marker?

Marker is a piece of
DNA molecule that is
associated with a certain
trait of a organism
 Types of Marker

Morphological

Types of
Biochemical Markers Genetic

Chromosomal
 Morphological Marker
•Phenotypic markers
•Naked eye marker

hulled naked Black white

 Morphological Markers
 Recessive in nature
 Mutations - deleterious phenotype

 Problems with epistasis, pleiotrophy, incomplete

penetrence
 Influenced by environment

 Transitory phenotype

 Difficult to combine
 Proteins Markers
Allozymes:
Isoenzymes of protein nature whose
synthesis is usually controlled by
codominant alleles and inherited by
monogenic ratios

Isozymes:
A species of enzyme that
exists into two or more
structural forms which are
easily identified by their
activities
 Common protein marker:
Isozymes
 Multiple forms of the same enzyme
- allozyme: one enzyme and one locus
- isozyme: one enzyme, more than one
locus (gene duplication; gene families)
 To be useful as markers, isoforms must be
electrophoretically resolvable, and detectable by
in-gel assay methods
 Methodology
 Grind and extract protein from appropriate
tissue with buffer.
 Fractionate extract electrophoretically in starch
or polyacrylamide gels (non-denaturing).
 Detect enzyme by incubation of gel (or gel print)
in a solution of a synthetic substrate that allows
the enzyme to catalyze a reaction that generates
a coloured product.
 Molecular Markers

Readily detectable sequence of protein or DNA that are closely

linked to a gene locus and/or a morphological or other characters
of a plant

Readily detectable sequence of protein or DNA whose inheritance

can be monitored and associated with the trait inheritance
independently from the environment

Types:
a) protein polymorphisms
b) DNA polymorphisms
 Molecular markers
 Molecular marker are based on naturally
occurring polymorphism in DNA sequence(i.e.
base pair deletion, substitution ,addition or
patterns).
 Genetic markers are sequences of DNA which
have been traced to specific locations on the
chromosomes and associated with particular
traits.
 It can be described as a variation that can be
observed.
 A genetic marker may be a short DNA
sequence, such as a sequence surrounding a
single base-pair change (single nucleotide
polymorphism, SNP), or a long one, like mini
satellites.
 Why use molecular markers
 A molecular marker is a DNA sequence which can be readily
detected and whose inheritance can be monitored.For example
in the case of pathogen resistance, a good marker avoids
elaborate infection tests for the selection of resistant plants.
 DNA-markers allow the breeder to introduce into their
cultivated plant only the gene(s) of interest from a related
species. While conventional breeding methods rely on the
transfer of the whole genome (along the gene of interest,
undesirable characters are also co-inherited and have to be
eliminated through back crossing followed by selection) DNA-
markers allow to eliminate in a few generations ‘undesired’
genome regions
 The introgression of recessive alleles through
classical back cross breeding is even more
lengthy as this requires additional generations of
selfing (to identify the homozygous recessive)
after every back-crossing
 Some characters like complex disease resistance
reaction or biotic stresses (QTLs) that show
continuous variation and do not fit into
Mendelian ratios are most difficult to detect and
transfer through conventional plant breeding,
due to interactions with the environment
How can molecular markers help?

Molecular markers allow working with genotype

information directly

Analyze the effect of the genotype on the

phenotype

Provide the breeder a tool to look into the ‘black

box’ of the genotype
 The ‚perfecT marker‘

 Polymorphic
 Reproducable

 Easy to use and economical

 High-throughput genotyping

 Automation

 Combination of different markers in one

reaction
 Characteristics of Ideal
Polymorphic Markers
 Co-dominant expression
 Nondestructive assay
 Complete penetrance
 Early onset of phenotypic expression
 High polymorphism
 Random distribution throughout the genome
 Assay can be automated
Co-dominant marker
Gel configuration Polymorphism
P1 P2 O1 O2 -Parent 1 : one band
-Parent 2 : a smaller band
-Offspring 1 : heterozygote = both bands
-Offspring 2 : homozygote parent 1

Dominant marker Polymorphism

Gel configuration Parent 1 : one band

P1 P2 O1 O2
-Parent 2 : no band
-Offspring 1 : homozygote parent 1
-Offspring 2 : ????
 Desirable properties
 Polymorphic
 Co-dominant inheritance
 Occurs throughout the genome
 Reproducible
 Easy, fast and cheap to detect
 Selectivity neutral
 High resolution with large number of samples
First Generation Markers
[Based on restriction fragment detection]

 RFLP
 RFLP - restriction fragment length
polymorphisms
 Isolate high quality DNA
 Digest with a combination of restriction
enzymes
 Fractionate digested samples by
electrophoresis
 Transfer fragments to membrane
 Hybridize with (non)radioactively labeled
DNA probe(s); detect by autoradiography
 RFLP (Restriction fragment length polymorphism)

RFLPs involves fragmenting a sample of DNA by a restriction

enzyme, which can recognize and cut DNA wherever a specific
short sequence occurs. A RFLP occurs when the length of a
detected fragment varies between individuals and can be used in
genetic analysis.

Advantages:
 Variant are co dominant

 Measure variation at the level of DNA sequence, not protein

sequence.
Disadvantage:
 Requires relatively large amount of DNA
RFLP techniques
RFLP Polymorphisms interpretation

MFG
1 1 2 3 4 5 6

6
 Advantages and disadvantages

• Advantages • Disadvantages
– Reproducible – Time consuming
– Co-dominant – Expensive
– Simple – Use of radioactive
probes
 Second Generation Markers
[Based on PCR]
PCR: Polymerase Chain Reaction
amplification of tracts of DNA defined
by border sequences that hybridize to
selected DNA polymerase primers
 Requires:
- target DNA
- thermostable DNA polymerase (Taq)
- oligonucleotide primer(s) (7-30nt)
- dNTPs + Mg++
PCR (Polymerase Chain Reaction)
 RAPD ( Random amplification of polymorphic DNA)

Random Amplification of Polymorphic DNA. It is a type of PCR

reaction, but the segments of DNA that are amplified are random.
Advantages:
 Fast

 Relatively inexpensive

 Highly variable

Disadvantage:
 Markers are dominant

 Presence of a band could mean the individual is either homozygous

or heterozygous for the Sequence - can’t tell which?
 Data analysis more complicated
RAPD: Random Amplified Polymorphic
DNA markers
(Williams et al., 1990 NAR 18, 6531)
 RAPD Polymorphisms among landraces of sorghum

Sequences of 10- Name Sequence

mer OP A08 5’ –GTGACGTAGG- 3’
RAPD primers OP A15 5’ –TTCCGAACCC- 3’
OP A 17
M 5’ –GACCGCTTGT- 3’
OP A19 5’ –CAAACGTCGG- 3’
OP D02 5’ –GGACCCAACC- 3’
RAPD gel configuration
Positivity:
 Anonymous markers - but can be converted
to SCARs (Sequenced Characterized Amplified
Region Markers)
 Fast, easy and cheap - commercial primer sets
available
 Low on time /labour
Negativity:
 Very sensitive.
 Good reproducibility but technically demanding.
 Relatively expensive technology.
 Bands are anonymous - interpretation of patterns
can be challenging.
 AFLP ( Amplified fragment length polymorphism)
In this analysis we can amplify restricted fragments and reduces the
complexity of material to be analyzed (approx 1000 folds).it can
be used for comparison b/w closely related species only.
Advantages:
 Fast

 Relatively inexpensive

 Highly variable

Disadvantage:
 Markers are dominant

 Presence of a band could mean the individual is either

homozygous or heterozygous for the Sequence - can’t tell which?
 AFLP Markers
Technically demanding
Reliable and stable
Moderate cost
Need to use different kits adapted to the
size of the genome being analyzed.
Like RAPD markers need to be converted
to quick and easy PCR based marker
AFLP: Amplified Fragment Length
Polymorphism
Positivity:
 Fast, easy and cheap - commercial primer sets
available
 Low on time /labour
 High reproducibility.
 Fractionate products on high resolution sequencing
gels.
Negativity:
 Very sensitive
 Good reproducibility but technically demanding
 Relatively expensive technology
 Bands are anonymous - interpretation of patterns
can be challenging
 Micro satellite polymorphism, SSR or Simple
sequence repeat
Microsatellites, Simple Sequence Repeats (SSRs), or Short
Tandem Repeats (STRs), are repeating sequences of 1-6 base
pairs of DNA.
Advantages:
 Highly variable

 Fast evolving

 Co dominant

Disadvantage:
 Relatively expensive and time consuming to develop
 SSR (Simple sequence repeat)
DNA markers which developed by amplifying microsatellite in
the genome

Sequence Primer
ACTGTCGACACACACACACACGCTAGCT (AC)7
TGACAGCTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACGCTAGCT (AC)8
TGACAGCTGTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACACACGCTAGCT (AC)10
TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACACACACACGCTAGCT (AC)12
TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA
 SSR polymorphisms

P1 AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG

P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG

P1 P2

Gel configuration
Simple Sequence Repeat
(microsatellite)
Example of application of
SSR marker
Positivity:
 Highly reproducible
 Low in time/labour

 Co-dominant

 Technically simple

Negativity:
 High cost of development
 Low multiplex ratio
 Features RFLP PCR- DFP RAPD Microsatellite SNP
RFLP
Detection method Hybridization PCR Hybridization PCR PCR PCR

Type of g DNA/ Sequence Mini satellite Arbitrarily Sequence Sequence

probe/primer cDNA sequence of specific synthetic design specific primers specific
used structural genes primers oligos primer primers
Requirement of Yes No/Yes Yes No/Yes No/Yes No/Yes
radioactivity

Extant of genomic Limited Limited Extensive Extensive Extensive Extensive

coverage

Degree of Low Low High Medium to High High

polymorphisms High

Phenotype Co dominant Co Co dominant Co Dominant Co

expression dominant dominant/D dominant
ominant

Possibility of No Yes No Yes Yes Yes

automation
 Gene mapping
 Pre and post natal diagnosis of
diseases
 Anthropological and molecular
evolution studies
 Molecular markers can be used for several
different applications including
 Germplasm characterization,
 Genetic diagnostics,
 Characterization of transformants,
 Study of genome
 Organization and phylogenic analysis.
 Paternity testing and the investigation of crimes.
 Measure the genomic response to selection in livestock