MOLECULAR MARKERS

UNIT 13A

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WHAT IS MARKER?

A molecular marker is a molecule contained within a sample taken

from an organism or other matter.

It can be used to reveal certain characteristics about the respective

source

Morphological

Types of
Biochemical Markers Genetic
•Landmarks on chromosomes that serve as reference points to the location
of other genes of interest when a genetic map is constructed.

Chromosomal 2
Animals are selected based on appearance

Eg. PIGMENTATION

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Animals are selected based on biochemical properties

Eg. Hb, AMYLASE, BLOOD GROUPS ETC.

Disadvantage:
Sex limited Three enzymes derived from pancreatic acinar cells—amylase, lipase, and
Age dependent the proenzyme trypsinogen—have been tested as biochemical markers of
acute pancreatitis (inflammation of the pancreas) ) serum amylase is the
Influenced by environment most commonly used of these in clinical practice.
It covers less than 10% of genome
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 Animals are selected based on structural & numerical variations

Eg. Structural and Numerical Variations

Structural- Deletions, Insertions etc.
Numerical- Trisomy, Monosomy, Nullysomy

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 In genetics, a molecular marker (identified as genetic marker) is a
fragment of DNA that is associated with a certain location within
the genome.

Genetic variation, in the form of multiple alleles of many genes,

occurs between organisms of a population and also within and
between natural populations
GENETIC
MARKERS Such genetic differences are due to DNA markers or (DNA
polymorphisms; poly = many, morph = form) that are present in the
genomes of organisms

Genetic (or DNA) markers are detected by direct analysis of the

DNA
➢ often, this requires genomic DNA that is fragmented into smaller
pieces that are easier to handle and manipulate to reveal genetic
differences, i.e. DNA sequence variation
 PROPERTIES OF A GOOD GENETIC MARKER
Polymorphic
➢ to allow the differentiation of chromosomes carrying the mutant gene from those carrying the normal gene
➢ polymorphisms are also used to measure genetic diversity

Reproducible – exchange of data between laboratories

Codominant inheritance – to allow the discrimination of homozygotes from heterozygotes

Evenly and frequently distributed throughout the entire genome

Discriminating – to allow the detection of genetic differences between closely related individuals

Not subjected to environmental influences/developmental stages

Inexpensive – easy, fast and cheap application

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 RESTRICTED FRAGMENT LENGTH POLYMORPHISM

• Botstein et al., (1980)

The first technology to enable the detection
of polymorphism at the DNA sequence
level

Based on DNA fragment length differences

after digesting genomic DNA with one or
more restriction enzymes and separated on
an agarose gel (Southern blot procedure is
followed)

• In RFLP, DNA polymorphism is detected

by hybridizing a chemically labelled DNA
probe to a Southern blot of DNA digested
by restriction endonucleases, resulting in
differential DNA fragment profile.
 RFLPS DISPLAY CODOMINANT INHERITANCE
Base changes lead to gaining of new sites
8 kb
Allele A

3 kb 5 kb
Allele B

Note: each arrow indicates a restriction site

Note: there is an internal restriction site for an enzyme only in allele B

Genotypes Fragments AA AB BB

AA 8 kb 8 kb
AB 3 kb, 5 kb, 8 kb
5 kb
BB 3 kb, 5 kb
3 kb

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 RFLP Mapping - Example
2 kb 3 kb 7 kb
RFLP Locus

Note: the restriction enzyme used always cuts the RFLP locus at the external sites but may or may not cleave the internal sites
(individual differences)

Note: each arrow indicates a restriction site

Consider: cut (+) and no cut (-)

+/+ +/- -/+ -/-

2 kb 2 kb 5 kb 12 kb
3 kb 10 kb 7 kb
7 kb
D B C A

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PCR BASED MARKERS

RAPD Dominant AFLP Dominant SCAR Dominant

SNP SSR
STS Codominant
Codominant Codominant
 Random Amplified Polymorphic DNA (RAPD)

RAPD (pronounced “rapid”) is a PCR-based molecular marker technique

❖ It uses single short oligonucleotide primers (8 – 10 bp) that are arbitrarily selected (i.e. random) to amplify a set
of DNA fragments distributed randomly throughout the genome
❖ Note: DNA amplification product is generated from a region that is flanked by a part of 10-bp priming sites
binding close together and in opposite directions
Genomic DNA from two different individuals often produce different amplification patterns (hence, Random
Amplified Polymorphic DNA)
A particular fragment generated for one individual but not the other represents DNA polymorphism and can be used
as a genetic marker – “genomic fingerprint”
A DNA sequence difference between individuals in a primer-binding site may result in the failure of the primer to
bind, hence no amplification
RAPDs display a dominant pattern of inheritance, i.e. the presence of a band indicates a dominant allele (e.g. AA
or Aa) whereas the absence of a band indicates a recessive allele (e.g. aa)

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 RAPD
1) For each RAPD polymorphism (i.e. phenotype),
let us call the “allele” capable for amplification
the plus (+) allele and the allele not capable of
amplification the minus (-) allele

2) Three possible genotypes, thus, +/+; +/-; and -/-

3) This means the homozygous (+/+) and

heterozygous (+/-) will both support
amplification, whereas the homozygous type (-/-
) will not support amplification

4) Hence, the presence of the amplified fragment is

the phenotype observed in both +/+ and +/- (with
the + allele being dominant over the - allele)

Note: a specific band occurring at a specific location in all individuals (A – D) in a specific

primer set is referred to as being monomorphic
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SEQUENCE CHARACTERIZED AMPLIFIED REGION (SCAR)
• SCARs are DNA fragments amplified by the PCR using specific 15-30 bp primers
• Designed from nucleotide sequences established from cloned RAPD fragments linked to a trait of interest.
• By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered with RAPDs.

Advantage:
1. Co-dominant inheritance
2. Amplify single locus
3. More informative than
RAPD
4. Stable and reproducible
5. No radioactivity
involved
SINGLE NUCLEOTIDE POLYMORPHISM (SNP)
SNP (pronounced “snip”) - polymorphisms caused by point mutations that give rise to different alleles containing
alternative bases at a given nucleotide position within a locus

An allele is a variant form of a given gene

They are the most abundant polymorphism in any organism.
For example:
➢ mouse – 1 SNP per 104 bp
➢ humans – 1 SNP per 1250 bp

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AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP)

AFLP is a PCR-based molecular marker technique

❖ Essentially a combination of RFLP & RAPD methods
❖ Based on the PCR amplification of genomic restriction fragments generated by specific restriction enzymes and
oligonucleotide adapters of few nucleotide bases
❖ It’s a DNA fingerprinting technique because it involves the display of a set of DNA fragments from a specific
DNA sample
❖ Fingerprints are produced without prior sequence knowledge, using a limited set of genetic primers

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• Genomic DNA digested with res. enzymes (generally two enzymes are
used, i.e. one rare cutter such as MseI and another a frequent cutter,
e.g. EcoRI)

• Ligate double-stranded oligonucleotide adapters to the ends of the

DNA fragments

• Selective amplification of restriction fragments using radioactively

labelled primers.

• Note: primers are complementary to adapters and may contain one or

more extra nucleotides at their 3’-end

• Gel analysis of the amplified fragments using highly resolving

sequencing gels and visualized using autoradiography

• After final amplification, selectively amplified fragments are

separated by gel electrophoresis and visualized autoradiographically.

• Typically, the autorad has 100-300 fingerprints with sizes ranging

from 80 to 500 nucleotides.

• Only a subset (10-40) of these total bands is polymorphic between

two related individuals, such as Arabidopsis thaliana
Columbia and Landsberg erecta ecotypes.
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AFLP Gel Electrophoresis - Example
Different experimental units, e.g. individuals

Monomorphic

Polymorphic

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 Type of DNA polymorphisms
resulting from differences in the
number of copies of a DNA Any chromosome may have any
sequence that may be repeated number of tandem repeats
many times in tandem at a
particular site in a chromosome
TANDEM REPEAT
POLYMORPHISMS
Note: duplex DNA containing
Number of copies may range
the repeats can also be
from 10 to a few hundred
amplified by means of PCR
SIMPLE SEQUENCE REPEATS (SSR)

Allele 1
Allele 2

Note: typical SSR has a repeat length of 2 – 9

nucleotides
Minisatellites are often referred to as VNTRs
Microsatellites are often referred to as Short Tandem Repeats (STRs) by forensic geneticists and in genetic genealogy, or as
Simple Sequence Repeats (SSRs) by plant geneticists. 26
 VARIABLE NUMBER
TANDEM REPEATS

Note: due to the larger repeating unit found

in VNTRs, they can easily be resolved by
Note: unlike SSRs, VNTRs have much
electrophoresis such as in DNA typing (or
longer repeating units, i.e. 10 – 60
DNA fingerprinting)
nucleotides

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Gene mapping

Pre and post natal diagnosis of diseases

Molecular evolution studies

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

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HOW IT IS
DONE?
• Restriction enzyme digestion of
DNA followed by Southern blot
analysis was used for DNA
fingerprinting.

https://www.blendspace.com/lessons/DMtRV_Zx3TfiGQ/dna-fingerprinting

DNA FINGERPRINTING
https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/dna-profiling.html
 MOLECULAR MARKER TECHNIQUES - COMPARISON
Characteristic RAPD RFLP AFLP SSRs (Microsatellites)

Principle of ID DNA amplification Restriction digestion DNA amplification DNA amplification

Principle of Analysis DNA staining Southern blotting DNA staining DNA staining

Primer requirement Yes (random) None Yes (selective) Yes (selective)

Probe requirement None Set of specific probes None None

Use of radioisotopes No Yes Yes/No Yes/No

Dominant/
Dominant /Codominant Dominant Codominant Codominant
Codominant
Polymorphism
Medium Medium Medium High
Detected

Reliability Intermediate High High High

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 Refers to the use of DNA markers that are tightly-linked to target
MARKER loci as a substitute for or to assist phenotypic screening

ASSISTED
SELECTION
(MAS) Assumption: DNA markers can reliably predict phenotype
ADVANTAGES OF MAS

• 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
• Increased reliability
• No environmental effects
• Can discriminate between homozygotes and heterozygotes and select single plants
Overview of
‘marker (1) LEAF TISSUE
genotyping’ SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

 • DNA molecular markers in plant breeding: current
status and recent advancements in genomic selection
and genome editing.
https://doi.org/10.1080/13102818.2017.1400401

• An overview of molecular marker methods for plants.

REFERENCES December 2006. AFRICAN JOURNAL OF
BIOTECHNOLOGY 525(25):2540-2568