1. Molecular markers are DNA sequences that can be used to identify specific locations or genes on chromosomes. Different types of molecular markers include isozymes, restriction fragment length polymorphisms (RFLPs), variable number of tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs). 2. Molecular markers have a variety of uses in genetics and forensic analysis. RFLPs and VNTRs can be used for DNA fingerprinting and identifying individuals. SNP markers allow for analysis of genetic variations between individuals and populations. Molecular markers are also used for genome mapping and marker-assisted breeding in plants and animals. 3. There are several techniques used to detect molecular markers, including gel electrophoresis, Southern blot

1. MOLECULAR MATKERS

2. NH 1
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s for
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visit
Just as mileposts guide the motorists along a linear highway, molecular
tools enable the geneticists to establish specific ‘DNA markers’ at
defined places along each chromosome. DNA markers can then be used
to delineate when one has reached or passed by a particular gene of

3. Molecular
MarkerA molecular marker is a polypeptide or piece of DNA with easily
identified phenotype such that cells or individuals with different
alleles are distinguishable. It can be a protein, isozyme, short DNA
sequence, such as a sequence surrounding a single base-pair change
(single nucleotide polymorphism, SNP), or a long one, like
minisatellites, whose inheritance can be monitored.
Ideal characteristics
• Polymorphic
• Reproducible
• Co-dominant
• Wide genome coverage
• Easy and inexpensive
• Easy exchange of data
1. Represent the genetic
difference
2. May or may not represent
the target gene
3. Occupy specific loci

4. Molecular
marker
Protein marker Hybridization
based marker
PCR based marker
•RFLP
•VNTR
•RAPD
•AFLP
•SSR
•ISSR
•SCAR
•CAPS
•EST
•Isozyme

5. Isozyme markers
Zymogram
Isozymes are proteins with same enzymatic function but
different structural, chemical, or immunological
characteristics.
Use: Population genetics, phylogeny, diversity,
Isozyme
Multiple forms of the
same enzyme coded
by the different genes,
one enzyme, more
than one locus.
Allozyme
One
enzyme;
one locus
Advantages: Easy and inexpensive; required no sequence
information; co-dominant
Disadvantages: Limited availability of enzyme system,
low level of polymorphism, environment and tissue
dependent

6. Restriction Fragment Length Polymorphism
Digestion with
restriction enzyme
Construct genomic or cDNA library
Develop probes
Electrophoresis
and Southern
blotting
Probe labelling
Hybridization
AA aa Aa
Marker scoring
Eco RI Eco RI
Eco RI Eco RI
Probe DNA Label
Labelled probe
Genomic
DNA

7. Restriction Fragment Length Polymorphism
Polymorphism: Point mutation, insertion, deletion or unequal
crossing over. It depends on enzyme probe combination.
Probes can be cDNA or PstI-derived genomic clone.
Advantages: Highly reproducible, co-dominant, wide genome
coverage, no sequence information, can be used across
species.
Disadvantages: Laborious, require high amount of DNA,
require radioactivity, difficult to automate.

8. Variable Number of Tandem Repeats (VN
Repetitive DNA
Majority of genome is constituted by tandem arrays of different
types of repetitive DNA, which play important roles in absorbing
mutation. Length polymorphisms arise due to polymerase slippage
during replication or unequal recombination.
Minisatellites
Tandem repeats with a monomer repeat length of about 11–60 bp
Microsatellites
Short tandem of 1 to 6 bp long monomer sequence
VNTR: Restriction digested genomic DNA is hybridized
with a particular minisatellite sequence.

9. Each variant acts as an inherited allele, allowing them to
be used for personal or parental identification. VNTRs
have become essential to forensic crime investigations,
via DNA fingerprinting and the CODIS database
Variable Number of Tandem Repeats (VN
The Combined DNA Index System (CODIS) is a DNA database funded by the United
States Federal Bureau of Investigation (FBI). It is a computer system that stores DNA
profiles created by federal, state, and local crime laboratories in the United States, with the
ability to search the database to assist in the identification of suspects in crimes

10. First cycle
Second cycle
Third cycle
Unit length RAPD products
Genomic DNA
Use one random decamer primer and amplify
by polymerase chain reaction
Electrophoresis
AA aa Aa
Marker scoring
40-45 cycles
Random
Amplified
Polymorphi
c DNA
(RAPD)

11. Random Amplified Polymorphic DNA
(RAPD)

12. Random Amplified Polymorphic DNA
(RAPD)
Multiple Arbitrary Amplicon Profiling (MAAP)
Collective term for techniques using single arbitrary primers
Use
Diversity, phylogeny
Advantage
Easy, inexpensive, no sequence information, low quantity of DNA, wide
genome coverage.
Disadvantage
Dominant, low reproducibility, sensitive to contamination
Arbitrarily Primed Polymerase Chain Reaction
(AP-PCR)
Uses longer arbitrary primers than RAPDs
DNA Amplification Fingerprinting (DAF)
Uses shorter, 5–8 bp primers

13. Electrophoresis
AA aa Aa
Marker scoring
Genomic DNA
Digestion with two
restriction enzymes
Ligation of adapter DNA
N
NN
Preselective-amplification
Use two selective primers
NNN
NN
Design longer selective primers
Selective PCR amplification
Amplified
Fragment
Length
Polymorphis
m (AFLP)

14. Uses
Genetic diversity, phylogeny, fingerprint & cultivar identification, contig
map, criminal & paternity test
Advantages
High polymorphism, wide genome coverage, low quantity of DNA,
amenable to automation
Disadvantages
Dominant, require good quality of DNA, use of radioactivity
AFLP fingerprint

15. Genomic DNA
P1
P2
Synthesize two primers per locus
from the flanking conserved regions
Polymerase Chain Reaction
Electrophoresis
AA aa Aa
Marker scoring
Simple Sequence Repeats
Di-nucleotide repeat:
CACACACA
Tri-nucleotide repeat:
ATGATGATGATG
Example of allelic variation in
SSRs:
Allele A: CACACACA (4 repeats of
the CA sequence)
Allele B: CACACACACACA (6
repeats of the CA sequence)
Microsatellites 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

16. Uses
Fingerprinting, genome mapping & linkage analysis, marker-assisted
selection
Advantages
Highly reproducible, co-dominant, wide genome coverage, low quantity
of DNA, PCR-based and nonradioactive, amenable to automation
Disadvantages
Need sequence information, can not be used across species
SSR fingerprint

17. Inter Simple Sequence Repeat (ISSR)

18. Sequence Tagged Site (STS)
PCR amplification of unique single copy segment
of the genome using long primer, designed based
on already available sequence information. So it
has the advantages of both RAPD (no probe,
PCR-based, rapidity) and RFLP (codominance,
highly reproducible).
Three types- SCAR, CAPS, STMS

19. Sequence Characterized Amplified Region
(SCAR)Select a unique RAPD fragment linked to a specific trait and
showing polymorphism
Gel cut the fragment, cloned into a suitable vector and end
sequence
Design primers based on the sequence information,
PCR amplification, gel run
High reproducibility as long sequence specific primers are
used, codominant inheritance, low quantity of DNA, quick
& easy, locus specific & can be used in gene mapping and
marker assisted selection (MAS). However, need
sequence information
Sequence Tagged Microsatellite Site (STMS): when microsatellite locus is ta

20. Cleaved Amplified Polymorphic Sequence
(CAPS)Amplify the target region using long PCR primer
Restriction digestion of the amplified fragment and gel run
High reproducibility as long sequence specific primers are
used, codominant inheritance, low quantity of DNA, quick
& easy, locus specific & can be used in gene mapping and
marker assisted selection (MAS). However, need
sequence information

21. SNPs are single base pair positions in genomic DNA, at which
different sequence alternatives (alleles) exist in normal
individuals in some population(s), herein the least frequent
allele has an abundance of 1% or greater.
Single Nucleotide Polymorphism
(SNP)

22. Several types of SNPs are distinguished, according to their
assignment to the structural element of genomic DNA or
their functional effect
Regulatory SNPs (rSNPs)
Involving regulatory regions that control gene expression (promoter SNPs
and some intron SNPs)
Anonymous SNPs
Functional effect is unknown
Candidate SNPs
Presumably having a functional effect
Protein SNPs
Change in the protein function or expression
Single Nucleotide Polymorphism
(SNP)

23. SNP
Genotyping
The detection of SNP markers on the basis of overlapping
genomic DNA sequences
The detection of SNP markers on the basis of overlapping EST
sequences
The detection of SNP markers on the basis of unique
(nonoverlapping) genomic and EST sequences
The detection of SNP markers on the basis of “shotgun”
sequencing

24. Allele
Discrimination
A C
T T
A
T
Match
C
T
Mismatch
Hybridization
No hybridization
Allele-specific hybridization

25. C
T
A
T
Match
C
T
Mismatch
Primer extension
No primer extension
Allele-specific PCR
A
T
Allele
Discrimination

26. C
T
A
T
Match
C
Mismatch
Nucleotide incorporation
No nucleotide incorporation
Allele-specific single-base primer extension
A
T
Allele
Discrimination

27. C
T
A
T
Match
C
Mismatch
Nucleotide incorporation
No nucleotide incorporation
Allele-specific single-base primer extension
A
T
Allele
Discrimination

28. Allele
Discrimination

29. The most straightforward way to detect an allele-
specific product is to label it by incorporating one or
more nucleotides conjugated to a fluorescent dye.
Direct fluorescence detection is generally used with
solid-phase assay formats (microarrays and bead
arrays) and where allele-specific products are
separated by gel electrophoresis or capillary
electrophoresis
Direct Fluorescence Detection

30. Fluorescence-based Detection in
Homogenous Solution

31. When a fluorophore is excited by plane-polarized light, the
fluorescence emitted by the dye is also polarized. This
phenomenon is termed fluorescence polarization (FP).
Complete FP occurs only when the dye molecule is
stationary. Therefore the degree of observed FP is
dependent on how fast a molecule tumbles in solution, and
this is in turn dependent on the volume of the molecule,
which is related to its molecular mass. Therefore changes
in molecular mass (e.g., caused by primer extension,
probe hydrolysis, or invasive cleavage) can be detected by
changes in FP as long as all other conditions (temperature,
viscosity, etc.) remain constant. FP is used as the
detection method in the SnaPshot (Applied Biosciences)
and Acycloprime (Perkin Elmer)
commercial genotyping systems.
Fluorescence Polarization

32. Signal detection is based on difference in molecular
weights of small DNA fragments rather than the
behavior of a label. The analysis of DNA by mass
spectrometry requires soft ionization (i.e., without
fragmentation) and is usually achieved by Matrix-
Assisted Laser Desorption/Ionization- Time of Flight
(MALDI-TOF) analysis. The MALDI procedure involves
mixing the allele-specific products of the
discrimination assay with a matrix compound on a
metal plate. The mixture is then heated with a short
laser pulse, causing it to expand into the gas phase
where ionization is achieved by applying a strong
potential difference. Ions are accelerated toward the
detector and the time of flight (the time taken to reach
the detector) is measured, allowing the mass/charge
Mass Spectrometry

33. Pyrosequencing is a novel method for sequencing short stretches of DNA based
on the detection of pyrophosphate, a normal by-product of DNA synthesis.
Although similar in principle to primer extension allele discrimination methods,
pyrosequencing is suitable not only for typing SNPs but also for scoring entire
haplotypes (groups of linked SNPs).
Pyrosequencing
A
T
PPi
Adenosine 5’ phosphosulfate (APS)
ATP sulfurylase
ATP
Luciferin
Oxyluciferin + Light
Luciferase

34. Genic Molecular Marker (GMM)
The markers, derived from genomic DNA, could belong to either the
transcribed or the non-transcribed part of the genome without any
information available on their functions. In contrast, GMMs are developed
from coding sequences like ESTs or fully characterized genes with known
functions.
Gene-targeted markers (GTMs)
Derived from polymorphisms within genes, however not necessarily
involved in phenotypic trait variation, e.g. untranslated regions (UTRs) of
EST sequences.
Functional markers (FMs)
Derived from polymorphic sequences or sites within genes and, thus, more
likely to be causally involved in phenotypic trait variation (e.g. candidate
gene-based molecular markers).
Indirect functional markers (IFMs): For which the role for phenotypic trait
variation is indirectly known, and Direct functional markers (DFMs): for
which the role for the phenotypic trait variation is well proven.

35. Genic Molecular Marker (GMM)
Expressed Sequence
Tag
Unigene Sequence Data
In silico mining
Software tools are
used to identify SSR,
SNP etc.
Direct mapping
Can be used as
RFLP probe or for
primer designing for
STS, CAPS etc.

36. DNA Fingerprinting
• Genetic variation
• Cultivar and strain identification
• Eco-geographical variation
• Mutation detection
• Selection of diverse parents
• Germplasm conservation

37. Genetic Variation
Similarity
0.56 0.66 0.76 0.85 0.95
K. Swarna
K. Giriraj
K. Chipsona2
K. Jeevan
K. Alankar
K. Khasigaro
K. Kumar
K. Megha
K. Jyoti
K. Muthu
K. Badshah
K. Sutlej
K. Chandramukhi
K. Dewa
K. Lauvkar
K. Ashoka
K. Red
K. Safed
K. Chamatkar
K. Jawahar
K. Bahar
K. Sheetman
K. Lalima
K. Kuber

38. Cultivar and Strain
Identification
OPC 05 OPC 06 OPC 09 OPD 05Descriptors
• Morphological
• Biochemical
• DNA based

39. Eco-Geographical Variation
Similarity Coefficient
0.50 0.61 0.72 0.84 0.95
Bokuta 7
Bokuta 9
Spilow 1
Dubling 5
Dubling 7
Spilow 8
Rispa 10
Kalpa 9
Kalpa 10
Kanan 10
Rispa 2
Moorang 1
Skiba 9
Shongthong 9
Kalpa 3
Kanan 2
Skiba 1
Shongthong 10
Dubling 10
Akpa 2
Akpa 10
Moorang 6
Skiba 3
Shongthong 1
Eco-geographical
variability in Pinus
gerardiana

40. Genome Mapping
Assigning/locating genes/markers to particular
region of a chromosome and determining the
location of and relative distances between
genes/markers on the chromosome
• There are two types of maps: genetic linkage map
and physical map
• The genetic linkage map shows the arrangement of
genes and genetic markers along the chromosomes
as calculated by the frequency with which they are
inherited together. The physical map is
representation of the chromosomes, providing the
physical distance between landmarks on the
chromosome, ideally measured in nucleotide bases.
The ultimate physical map is the complete

41. Genetic Linkage Map
The three main steps of linkage map
construction are:
(1)Production of a mapping population
(2)Identification of polymorphism
(3)Linkage analysis of markers

42. Development of Mapping
Population

43. Marker A
Marker C
Marker D
A
B
B
C
C
D
D
14 6 4
…. 10 12
…. …. 2
…. …. ….
A D C
4/20 2/2
0
6/20
Recurrentparent
Donorparent
F1hybridMarker B
Linkage Analysis

44. Linkage Map

45. Comparative Mapping or
Synteny• The grass model
• Tomato- potato co-linearity
• Map based prediction of location of
orthologous genes
Cross mapping of DNA
probes in a range of cereal
crops has led to the
widespread identification of
chromosomal regions in
which marker orders are
highly conserved. It is now
possible to describe all the
genomes of grass crop
species by their relationship
to a single reference genome,
rice. Potato genomes were mapped into 12 linkage groups
corresponding to 12 potato chromosomes using tomato cDNA
clones. Three paracentric inversions in chromosomes 5, 9, 10 were
detected in relation to the tomato chromosomes. The other nine

46. Species kilobases/centimorgan
Arabidopsis 139
Tomato 510
Corn 2140
Physical and genetic distances are resolved by hybridizing clones, which
define closely linked genetic markers, to DNA that has been cut with rare
cutting enzymes. If two clones hybridize to the same fragment, then the
maximum distance between those two clones is the size of the restriction
fragment. As the genetic distance between the loci is known, genetic and
physical distances in this region can be correlated.
Physical
Mapping
The correspondence between genetic and physical distance
varies widely at different locations within a genome due to
recombination suppressed or hotspot regions

47. Fluorescence in situ Hybridization
(FISH)
Metaphase cell
Array on glass slide
Hybridize with labelled probe
Fluorescent
counterstaining of
chromosome
Visualization on
fluorescent microscope

48. Marker-assisted
Selection
MAS refers to the use of DNA markers
that are tightly-linked to target loci as
a substitute for or to assist
phenotypic screening
Assumption: DNA markers can reliably
predict phenotype

49. High-resolution Linkage
Mapping

50. • Markers must be tightly-linked to target loci.
• Ideally markers should be <5 cM from a gene or QTL
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM 5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%
• Using a pair of flanking markers can greatly improve reliability but
increases time and cost
Marker-assisted
Selection

51. Markers should be validated by testing their
effectiveness in determining the target
phenotype in independent populations and
different genetic backgrounds, which is referred
to as ‘marker validation’. In other words, marker
validation involves testing the reliability of
markers to predict phenotype. This indicates
whether or not a marker could be used in
routine screening for MAS.
Validation of
Markers

52. Marker
Conversion
There are two instances where markers may
need to be converted into other types of
markers: when there are problems of
reproducibility (e.g. RAPDs) and when the
marker technique is complicated, time-
consuming or expensive (e.g. RFLPs or
AFLPs). The problem of reproducibility may be
overcome by the development. of sequence
characterised amplified regions (SCARs) or
sequence-tagged sites (STSs) derived by
cloning and sequencing specific RAPD
markers.

53. F2
P2
F1
P1 x
large populations consisting of thousands
of plants
ResistantSusceptible
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers
Marker-assisted
Selection

54. • Simpler method compared to
phenotypic screening
– Especially for traits with laborious screening
– May save time and resources
• Selection at seedling stage
– Important for traits such as grain quality
– Can select before transplanting in rice
• Increased reliability
– No environmental effects
– Can discriminate between homozygotes and
heterozygotes and select single plants
Marker-assisted
Selection

55. • more accurate and efficient selection
of specific genotypes
– May lead to accelerated variety
development
• more efficient use of resources
– Especially field trials
Marker-assisted
Selection

56. Map Based Cloning
• Marker targeting
• Chromosome
walking
Chromosome

57. Bulked Segregants Analysis
(BSA)

58. A BGen
e
Chromosome Walking

59. Chromosome Landing
A B CGen
e
0.78 cM
0.16 cM 11.3 cM
B D E F
Gene

60. Target Gene Identification
RFLP B RFLP G RFLP D RFLP H RFLP E
Gene

61. • High-resolution mapping to demonstrate cosegregation of
the candidate clone with the phenotype
• Demonstration that the expression pattern of the gene is
consistent with the phenotype, for example, that it is
transcribed in the appropriate tissue(s) and/or induced by
the appropriate stimulus
• Determination of the DNA sequence of the gene and its
comparison with those in sequence databases to identify
any homology with genes of known function
• Probing mutant lines with the cDNAs to identify alterations
of the DNA or mRNA from the wild type
• Complementation of the mutant phenotype by
transformation with the gene
Target Gene Validation