A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.

1. Molecular marker technology in studies on plant
genetic diversity
PCR based
techniques
PCR with arbitrary
primers
PCR with
Amplified
fragment
length
polymorphis
ms
Sequence-
tagged sites
Microsatellit
es,
SCARs,
CAPS,
ISSRsBy
Chanakya
Pachi

2. Molecular Markers
• The development of molecular techniques for genetic
analysis has led to a great augmentation in our
knowledge of crop genetics and our understanding of
the structure and behavior of various crop genomes.
• These molecular techniques, in particular the
applications of molecular markers, have been used to
scrutinize DNA sequence and it’s variation(s) in and
among the crop species and create new sources of
genetic variation by introducing new and favorable
traits from landraces and related crop species.

3. Markers can aid selection
for target alleles that
• Are not easily assayed in
individual plants,
• Minimize linkage drag
around the target gene, and
• Reduce the number of
generations required to
recover a very high
percentage of the recurrent
parent genetic background

4. Introduction
• A genetic marker is a gene or DNA sequence with a known
location on a chromosome and associated with a particular gene
or trait.
• It can be described as a variation, which may arise due to
mutation or alteration in the genomic loci, 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 minisatellites.

5. Some
commonly
used types of
genetic
markers
RFLP (or
Restriction
fragment length
polymorphism)
AFLP (or
Amplified
fragment length
polymorphism
RAPD (or
Random
amplification of
polymorphic
DNA)
VNTR (or
Variable number
tandem repeat)
Microsatellite
polymorphism SNP (or Single
nucleotide
polymorphism)
STR (or Short
tandem repeat)
SFP (or Single
feature
polymorphism)
DArT (or
Diversity Arrays
Technology)

6. DNA-based technologies
• PCR based techniques
• PCR with arbitrary primers
• Amplified fragment length polymorphisms
(AFLPs)
• Sequence-tagged sites
• Microsatellites
• SCARs
• CAPS
• ISSRs

7. PCR BASICS
• The polymerase chain reaction
• PCR is a rapid, inexpensive and simple way of
copying specific DNA fragments from minute
quantities of source DNA material
• It does not necessarily require the use of radioisotopes or
toxic chemicals
• It involves preparation the sample DNA
• A master mix with primers,
• Followed by detecting reaction products

8. PCR procedures: steps
• Denaturation:
• DNA fragments are heated at high temperatures, which
reduce the DNA double helix to single strands.
• These strands become accessible to primers
• Annealing:
• The reaction mixture is cooled down.
• Primers anneal to the complementary regions in the DNA
template strands, and double strands are formed again
between primers and complementary sequences
• Extension:
• The DNA polymerase synthesises a complementary strand.
• The enzyme reads the opposing strand sequence and
extends the primers by adding nucleotides in the order in
which they can pair.

9. • The DNA polymerase, known as 'Taq polymerase', is named after
the hot-spring bacterium Thermus aquaticus from which it was
originally isolated.
• The enzyme can withstand the high temperatures needed for
DNA-strand separation, and can be left in the reaction tube.
• The cycle of heating and cooling is repeated over and over,
stimulating the primers to bind to the original sequences and to
newly synthesised sequences.
• The enzyme will again extend primer sequences.
• This cycling of temperatures results in copying and then copying
of copies, and so on, leading to an exponential increase in the
number of copies of specific sequences.
• Because the amount of DNA placed in the tube at the beginning is
very small, almost all the DNA at the end of the reaction cycles is
copied sequences. The reaction products are separated by gel
electrophoresis.
• Depending on the quantity produced and the size of the amplified
fragment, the reaction products can be visualized directly by
staining with ethidium bromide or a silver-staining protocol, or by

10. PCR procedures: cycle 1

11. PCR procedures: cycle 2

12. PCR procedures: cycle 3

13. CONDITIONS
Step Reaction Temperature (°C) Time (Seconds)
1 Initial Denaturation 94 120
2 Cycle Denaturation 94 15
3 Cycle Annealing 58 30
4 Cycle Extension 72 80
5 Repeat from Step 2 to 4 30 Cycles
6 Final extension 72 600
7 Infinite Hold 4

14. PCR with arbitrary primers
• MAAP (multiple arbitrary amplicon profiling) is
the acronym proposed to cover the three main
technologies that fall in this category:
• Random amplified polymorphic DNA (RAPD)
• DNA amplification fingerprinting (DAF)
• Arbitrarily primed polymerase chain reaction
(AP-PCR)

15. RAPD
• Random Amplified Polymorphic DNA
• It is a type of PCR reaction, but the segments of DNA that
gets amplified are random.
• The researcher performing RAPD creates several
arbitrary, short primers (8–12 nucleotides)
• Then proceeds with the PCR using a large template of
genomic DNA, to enable amplification of fragments.
• By resolving the resulting patterns, a semi-unique profile
can be gleaned from a RAPD reaction.
• No knowledge of the DNA sequence for the targeted
genome is required, as the primers will bind somewhere
in the sequence, but it is not certain exactly where.

16. • This makes the method popular for comparing the DNA of biological
systems that have not had the privileged attention of the scientific
community, or in a system in which relatively few DNA sequences are
compared (it is not suitable for forming a DNA databank).
• Because it relies on a large, intact DNA template sequence, it has some
limitations in the use of degraded DNA samples.
• Its resolving power is much lower than targeted, species specific DNA
comparison methods, such as short tandem repeats.
• In recent years, RAPD has been used to characterize, and trace, the
phylogeny of diverse plant and animal species.

17. How it Works?
• Unlike traditional PCR analysis, RAPD does not
require any specific knowledge of the DNA
sequence of the target organism:
• The identical 1somer primers may or may not
amplify a segment of DNA, depending on positions
that are complementary to the primers' sequence.
• For example, no fragment is produced if primers
annealed too far apart.
• Therefore, if a mutation has occurred in the
template DNA at the site that was previously
complementary to the primer, a PCR product will
not be produced, resulting in a different pattern of
amplified DNA segments on the gel

18. Diagrammatic summary

19. • This picture shows an image of a very high quality RAPD gel. Both,
presence and absence of most bands are very clear and the background
is transparent.
• The researcher would have no doubts while selecting bands and
collecting data from this gel.
• Consequently, the interpretation of results can be very confident.

20. Interpreting RAPD banding
patterns
• DNA polymorphism among individuals can be
because of:
• Mismatches at the primer site
• The appearance of a new primer site
• The length of the amplified region between primer sites
• Because of the nature of RAPD markers, only the
presence or absence of a particular band can be
assessed.
• Criteria for selecting scoring bands:
• Reproducibility—need to repeat experiments
• Thickness
• Size

21. Examples
Bell
pepper
Modern
rose
Meadow
fescue

22. Amplified fragment length
polymorphisms(AFLPs)
• AFLP is a PCR-based tool used in genetics research, DNA
fingerprinting, and in the practice of genetic engineering.
• Developed in the early 1990s by Keygene
• AFLP uses restriction enzymes to digest genomic DNA,
followed by ligation of adaptors to the sticky ends of the
restriction fragments.
• A subset of the restriction fragments is then selected to be
amplified.
• This selection is achieved by using primers complementary to
the adaptor sequence, the restriction site sequence and a few
nucleotides inside the restriction site fragments.
• The amplified fragments are separated and visualized on
polyacrylamide gels, either through autoradiography or
fluorescence methodologies, or via automated capillary

23. Digestion
Amplificatio
n
Electrophore
sis
AFLP
The procedure of this technique is divided into three steps:
• Digestion of total cellular DNA with one or more restriction
enzymes and ligation of restriction halfsite specific adaptors to all
restriction fragments.
• Selective amplification of some of these fragments with two PCR
primers that have corresponding adaptor and restriction site
specific sequences.
• Electrophoretic separation of amplicons on a gel matrix, followed
by visualisation of the band pattern.

24. Main features:
• A combination of the RFLP and PCR technologies
• Based on selective PCR amplification of restriction fragments
from digested DNA
• Highly sensitive method for fingerprinting DNA of any origin
and complexity
• Can be performed with total genomic DNA or with cDNA
('transcript profiling')
• DNA is digested with two different restriction enzymes
• Oligonucleotide adapters are ligated to the ends of the DNA
fragments
• Specific subsets of DNA digestion products are amplified, using
combinations of selective primers
• Polymorphism detection is possible with radioisotopes,
fluorescent dyes or silver staining

25. DNA digestion and ligation
• One restriction enzyme is a frequent cutter (four-
base recognition site, e.g. MseI)
• The second restriction enzyme is a rare cutter (six-
base recognition site, e.g. EcoRI)
• Specific synthetic double-stranded adapters for
each restriction site are ligated to the DNA
fragments generated

26. Summarizing the technology

27. Interpreting AFLP bands
• The AFLP technique detects polymorphisms arising from
changes (presence or size) in the restriction sites or
adjacent to these
• Different restriction enzymes can be used, and different
combinations of pre- and selective nucleotides will
increase the probability of finding useful polymorphisms
• The more selective bases, the less polymorphism will be
detected
• Bands are usually scored as either present or absent
• Heterozygous versus homozygous bands may be detected,
based on the thickness of the signal, although this can be
tricky

28. Applications
• Genetic diversity
assessment
• Genetic distance
analysis
• Genetic fingerprinting
• Analysis of germplasm
collections
• Genome mapping
• Monitoring diagnostic
markers
Examples
• Limonium sp.
• Stylosanthes sp.
• Indian mustard

29. PCR-based technologies
Sequences-tagged sites
Microsatellites SCARs CAPS ISSRs

30. Unlike arbitrary primers, STS
rely on some degree of
sequence knowledge
Markers based on STS are
codominant
They tend to be more
reproducible because
longer primer sequences are
usedRequire the same basic
laboratory protocols and
equipment as standard PCR
Sequence-tagged sites as markers

31. Microsatellites
• Microsatellites are also called simple sequence repeats
(SSRs)
• Microsatellites are short tandem repeats (1-10 bp)
• To be used as markers, their location in the genome of
interest must first be identified
• Polymorphisms in the repeat region can be detected by
performing a PCR with primers designed from the
DNA flanking region

32. Identifying microsatellite regions

33. Structure

34. Selecting primers

35. Methodology and visualisation
• Methodology:
• DNA extraction
• PCR with primers specific for microsatellite flanking
regions
• Separation of fragments
• Visualization:
• By agarose gel electrophoresis, using ethidium bromide
staining and UV light, or
• Acrylamide gels using silver staining or radioisotopes,
or
• Through automated sequencers, using primers
prelabelled with fluorescence.
• Data analysis

36. Staining with Ethidium bromide
Staining with Silver Nitrate

37. • Advantages:
• Require very little and not necessarily high quality DNA
• Highly polymorphic
• Evenly distributed throughout the genome
• Simple interpretation of results
• Easily automated, allowing multiplexing
• Good analytical resolution and high reproducibility
• Disadvantages:
• Complex discovery procedure
• Costly

38. Applications:
Individual genotyping
Germplasm evaluation
Genetic diversity
Genome mapping
Phylogenetic studies
Evolutionary studies

39. Sequence characterized amplified
regions (SCARs)
• SCARs take advantage of a band generated through a RAPD
experiment
• They use 16-24 bp primers designed from the ends of cloned
RAPD markers
• This technique converts a band—prone to difficulties in
interpretation and/or reproducibility—into being a very
reliable marker

40. Steps to obtain SCAR
polymorphismsA potentially
interesting band is
identified in a RAPD
gel
The band is cut out of the gel
The DNA fragment is
cloned in a vector and
sequenced
Specific primers
(16-24 bp long)
for that DNA
fragment are
designed
Re-amplification of the
template DNA with the new
primers will show a new and

41. Diagram of the SCAR procedure

42. Advantages Disadvantages

43. Cleaved amplified polymorphic
sequence (CAPS)
• This method is based on
• The design of specific primers,
• Amplification of DNA fragments, and
• Generation of smaller, possibly variable, fragments by
means of a restriction enzyme
• This technique aims to convert an amplified band
that does not show variation into a polymorphic one

44. Steps for generating CAPS
A band, DNA, gene sequence
or other type of marker is
identified as important
Either the band is detected
through PCR (and cut out of
the gel, and the fragment
cloned and sequenced) or the
fragment sequence is already
available
Specific primers are designed
from the fragment
sequence
The newly designed primers
are used to amplify the
template DNA
The PCR product is subjected
to digestion by a panel of
restriction enzymes
Polymorphism may be
identified with some of the
enzymes

45. Generating CAPS

47. Inter-simple sequence repeats
(ISSRs)
• They are regions found between microsatellite
repeats
• The technique is based on PCR amplification of
intermicrosatellite sequences
• Because of the known abundance of repeat
sequences spread all over the genome, it targets
multiple loci

48. Identifying ISSR polymorphisms
• A typical PCR is performed in which primers have
been designed, based on a microsatellite repeat
sequence, and extended one to several bases into
the flanking sequence as anchor points.
• Different alternatives are possible:
• Only one primer is used
• Two primers of similar characteristics are used
• Combinations of a microsatellite-sequence anchored
primer with a random primer (i.e. those used for RAPD)

49. Designing primers for ISSR
polymorphisms

50. • The diagram above presents three different items:
• The original DNA sequence in which two different repeated
sequences (CA), inversely oriented, are identified. Both
repeated sections are, in addition, closely spaced.
• If primers were designed from within the repeated region
only, the interrepeat section would be amplified but locus-
specificity might not be guaranteed. In the second row, a PCR
product is shown as a result of amplification from a 3'-
anchored primer (CA)n NN at each end of the interrepeat
region. CA is the repeat sequence that was extended by NN,
two nucleotides running into the interrepeat region.
• Alternatively, anchors may be chosen from the 5' region. The
PCR product in the third row is a result of using primers
based on the CA repeat but extended at the 5' end by NNN and
NN, respectively

51. • Do not require prior sequence
information
• Variation within unique regions of the
genome may be found at several loci
simultaneously
• Tend to identify significant levels of
variation
• Microsatellite sequence-specific
• Very useful for DNA profiling, especially
for closely related species
Disadvantages
• Dominant markers
• Polyacrylamide gel electrophoresis and
detection with silver staining or
radioisotopes may be needed

52. Latest strategies
DNA sequencing
Expressed sequence tags
(ESTs)
Microarray technology
Diversity array technology
(DArT)
Single nucleotide
polymorphisms (SNPs)

53. References…
References
Plant
Biotechnology
and Molecular
Markers
• P.S. Srivastava
• Alka Narula
• Sheela Srivastava
Using molecular
marker
technology in
studies on plant
genetic diversity
• M. Carmen de
Vicente
• Theresa Fulton
Molecular
Markers
• Ashwani Kumar
• Scientific
Blogging 2.0