This document discusses various biochemical and molecular markers that can be used for plant genetic characterization. It begins by explaining that genetic resources can be characterized based on genotypes, phenotypes, and various molecular traits. It then discusses different types of molecular markers including DNA fingerprinting using microsatellites, RAPDs, AFLPs, allozymes, and other protein markers. It provides details on how many of these techniques work, such as the PCR process for RAPDs and AFLPs. The document also discusses using these molecular markers for assessing population structure, assembling core collections, and addressing various taxonomic and phylogenetic questions.

1. BIOCHEMICAL AND MOLECULAR
MARKERS FOR
CHARACTERIZATION
T.MITHRAA
2016601602

2. Characterization
• Genetic resources can be characterized with respect to
genotypes, phenotypes - morphological traits, agronomic,
biochemical, cytological, molecular .
• Measures of genetic diversity, genetic distance, population
size and structure, geographical distribution, and degree of
endangerment

3. Characterization
• DNA fingerprinting / Molecular diversity
studies
• Assessing population structure
• Assembly of core collections
• Biogeographical, taxonomic and ecological
issues

4. DNA fingerprinting/Molecular
genetic diversity
• To identify genetically similar or distinct accessions, and to determine
individual degrees of heterozygosity and heterogeneity within
populations
• In Lycopersicon, microsatellites analysis revealed high level of
polymorphism within and among species, which was highly correlated
with the respective mating systems, cross-pollinating species having a
significantly higher gene diversity compared to self-pollinating species
(Alvarez et al., 2001).
• Molecular markers have been employed for genetic diversity studies in
many crop species Rice, Sorghum, Wheat, Barley, Castor, Sunflower
etc,

5. DNA fingerprinting/Molecular genetic
diversity
Marker analysis of a threatened common bean landrace
showed that in situ conservation is the most effective way to
maintain the diversity (Negri and Tiranti, 2010)
In capsicum, novel genetic variation was found in both the in
situ population studied and in some ex situ accessions, thus
supporting conservation of this species via both strategies
(Votava et al., 2002).
The genetic diversity of maize populations maintained in situ
and ex situ was found to be substantially equal (Rice et al.,
2006).

6. Taxonomic and phylogeny analysis
Taxonomic relationship between Vitis vinifera and Cayratia
saponaria using microsatellites found sufficient interspecific
variation to distinguish the two very closely related species.
Rossetto et al. (2002)
Phylogenetic analysis on domesticated and wild germplasm
accessions of sunflower using microsatellite loci, which
uncovered the possibility of multiple domestication origin
in sunflower. Tang and Knapp (2003)

7. Assembly of Core collection
• Markers have generated genetic diversity information to assist in
the establishment of core collections for many species, including
maize (Franco et al., 2006; Qi-Lun et al., 2008), wheat (Hao et al.,
2006), rice (Ebana et al., 2008), potato (Ghislain et al., 2006),
chick pea (Upadhyaya et al.,2008), grape (Le Cunff et al., 2008),
cacao and pepper (Marita et al., 2000).
• For creating core collections, emphasis should be placed on
methodologies that use data generated by markers in concert
with the morphological and agronomical characterization of the
accessions.
• Molecular markers have been used to develop thematic core
collections that are much smaller in size but exhibit the
maximum allelic richness for specific traits of interest (Upadhaya
et al., 2010).

8. BIOCHEMICAL MARKERS
• Monoterpenes
• Monoterpenes are a subgroup of the terpenoid substances found in
resins and essential oils of plants (Kozlowski and Pallardy, 1979).
• Although the metabolic functions of monoterpenes are not fully
understood, they probably play an important role in resistance to
attack by diseases and insects (Hanover, 1992).
• The concentrations of different monoterpenes, such as alpha-
pinene, beta-pinene, myrcene, 3-carene and limonene are
determined by gas chromotography and are useful as genetic
markers (Hanover, 1966a, b, 1992; Squillace, 1971; Strauss and
Critchfield, 1982).

9. • Monoterpene genetic markers have been applied primarily to
taxonomic and evolutionary studies.
• However, they have also been used to a limited extent to estimate
genetic patterns of geographic variation within species.
• Although monoterpenes were the best available genetic markers
for forest trees in the 1960s and early 1970s, they require
specialized and expensive equipment for assay.
• Monoterpenes were gradually replaced by allozyme genetic
markers because allozymes are less expensive to apply, are
codominant in expression, and many more marker loci can be
assayed.

10. Allozymes
• Allozymes have been the most important type of genetic marker in
forestry and are used in many species for many different
applications (Conkle, 1981; Adams et al., 1992).
• Allozymes are allelic forms of enzymes that can be distinguished by
a procedure called electrophoresis.
• The more general term for allozymes is isozymes, and refers to any
variant form of an enzyme, whereas allozyme implies a genetic
basis for the variant form.
• Most allozyme genetic markers have been derived from enzymes of
intermediary metabolism, such as enzymes in the glycolytic
pathway; however, conceivably an allozyme genetic marker could
be developed from any enzyme.

12. • Allozyme analysis : (a) Step 1, tissues for assay are prepared such as
megagametophytes and embryos from conifer seeds;
• Step 2, tissues are homogenized in an extraction buffer and absorbed onto small
filter paper wicks;
• Step 3, wicks are loaded onto gels and samples are electrophoresed;
• Step 4, gel slices are stained to reveal allozyme bands; and (b) Three general types
of allozyme patterns are observed in heterozygotes carrying slow and fast
migrating variants, depending on whether an enzyme is functional as a monomer
(lane 1), dimer (lane 2) or multimer (lane 3).
• Note that in all three cases, both alleles are expressed (i.e. are codominant) in
heterozygotes.

13. Other Protein Markers
• Another type of protein-based genetic marker utilizes two-dimensional
polyacrylamide gel electrophoresis (2-D PAGE).
• Unlike allozymes where single known enzymes are assayed individually,
the 2-D PAGE technique simultaneously reveals all enzymes and other
proteins present in the sample preparation.
• The proteins are revealed as spots on gels and marker polymorphisms are
detected as presence or absence of spots. This technique has been used
most extensively for linkage mapping in Pinus pinaster where protein
polymorphisms have been assayed from both seed and needle tissues
(Bahrman and Damerval, 1989; Gerber et al., 1993; Plomion et al., 1997).
• Although 2-D PAGE has the potential advantage that many marker loci
can be assayed simultaneously on a single gel, assays are more difficult
than in allozyme analyses, and the markers are often dominant in their
expression.

14. MOLECULAR MARKERS

15. 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.

16. 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.

17. 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)

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

19. 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

20. 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.
– The whole process is repeated over and over

21. • 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 means of radioisotopes and autoradiography

22. PCR procedures: cycle 1

23. PCR procedures: cycle 2

24. PCR procedures: cycle 3

25. 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

26. PCR based
techniques
PCR with arbitrary
primers
PCR with
Amplified
fragment
length
polymorphis
ms
Sequence-
tagged sites
Microsatellit
es,
SCARs,
CAPS,
ISSRs

27. 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)

28. 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.

29. • 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.

30. 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

31. • 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.

32. 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
– Expected segregation observed in a mapping population

33. 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, th
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
sequencing instruments

34. 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.

35. 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

36. 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

37. Summarizing the technology

38. 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

39. 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

40. 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
used
Require the same basic
laboratory protocols and
equipment as standard PCR
Sequence-tagged sites as markers

41. 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

42. Identifying microsatellite regions

43. Structure

44. Selecting primers

45. 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

46. Staining with Ethidium bromide
Staining with Silver Nitrate

47. • 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

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

49. 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

50. Steps to obtain SCAR
polymorphisms
A 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

51. Diagram of the SCAR
procedure

52. Advantages Disadvantages

53. 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

54. 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

55. Generating CAPS

57. 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

58. 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)

59. Designing primers for ISSR
polymorphisms

60. • 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

61. • 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

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