Biological markers

1. Genetic Markers

2. Variation
The differences that distinguish one individual
from another are encoded in the individual’s
genetic material, the deoxyribonucleic acid
(DNA). DNA is packaged in chromosome pairs,
one coming from each parent. The genes, which
control a plant’s characteristics, are located on
specific segments of each chromosome.

3. Marker

5. Genetic markers
Genetic markers are the biological features that
are determined by allelic forms of genes or
genetic loci and can be transmitted from one
generation to another, and thus they can be
used as experimental probes or tags to keep
track of an individual, a tissue, a cell, a nucleus,
a chromosome or a gene.

6. Genetic Markers
• represent genetic differences between individual
organisms or species
• do not represent the target genes themselves but act as
‘signs’ or ‘flags
• located in close proximity to genes (i.e. tightly linked)
may be referred to as gene ‘tags
• do not affect the phenotype of the trait of interest
because they are located only near or ‘linked’ to genes
controlling the trait
• occupy specific genomic positions within chromosomes
(like genes) called ‘loci’ (singular ‘locus’)

7. Classification of Genetic Markers
• Classical markers
• Morphological markers,
• Cytological markers
• Biochemical markers
• DNA markers
• RFLP, AFLP, RAPD, SSR, SNP

8. Morphological markers:
• visible traits, such as leaf shape, flower color,
pod color, seed color, seed shape, awn type
and length, fruit shape, stem length
• Some of these markers are linked with other
agronomic traits and thus can be used as
indirect selection criteria in practical breeding

10. Example of morphological markers
• Mendelian characters
• In wheat breeding, the dwarfism governed by
gene Rht10 was introgressed into Taigu
nuclear male-sterile wheat by backcrossing
and a tight linkage was generated between
Rht10 and the male-sterility gene Ta1
• Then the dwarfism was used as the marker for
identification and selection of the male-sterile
plants in breeding populations

11. Drawbacks of morphological markers
• Limited in numbers
• many of these markers are not associated with
important economic traits (e.g. yield and
quality)
• influenced by environmental factors or the
developmental stage of the plant
• However, despite these limitations,
morphological markers have been extremely
useful to plant breeders

12. Cytological markers:
• In cytology, the structural features of
chromosomes can be shown by chromosome
karyotype and bands. The banding patterns,
displayed in color, width, order and position,
reveal the difference in distributions of
euchromatin and heterochromatin

13. Biochemical/protein markers:
• Isozymes are alternative forms or structural
variants of an enzyme that have different
molecular weights and electrophoretic
mobility but have the same catalytic activity or
function. Isozymes reflect the products of
different alleles rather than different genes
because the difference in electrophoretic
mobility is caused by point mutation as a
result of amino acid substitution

14. Drawbacks of biochemical markers
• Limited in numbers
• influenced by environmental factors or the
developmental stage of the plant
• However, despite these limitations,
biochemical markers have been extremely
useful to plant breeders

15. Molecular/DNA markers
DNA markers are defined as a fragment of DNA revealing
mutations/variations, which can be used to detect
polymorphism between different genotypes or alleles of
a gene for a particular sequence of DNA in a population
or gene pool. Such fragments are associated with a
certain location within the genome and may be detected
by means of certain molecular technology.
DNA marker is a small region of DNA sequence showing
polymorphism (base deletion, insertion and substitution)
between different individuals

18. Defined
• RFLPs are differences in restriction fragment
lengths caused by SNPs or INDELs that create or
abolish restriction endonuclease recognition sites.
RFLP assays are performed by hybridizing a
chemically labelled DNA probe to a Southern blot of
DNA digested with a restriction endonuclease.
Restriction Fragment Length Polymorphisms
(RFLPs)

19. RFLPs
• Restriction fragment
length polymorphism
• Co-dominant
• Requires:
single copy DNA probe
Restriction enzyme
Southern blotting
DNA polymorphism

20. RAPDs
• Randomly
amplified
polymorphic DNA
• Based on a 10 bp
single arbitrary
primer
• Cheap, easy
• Insufficient
reproducible maize lines; only primer 2 and 5
demonstrate polymorphism

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RAPD – dominant marker
A
B

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AFLP: Major Steps
• Restriction endonuclease digestion of genomic
DNA and ligation of specific adapters
• Amplification of the restriction fragments by PCR
using primer pairs containing common sequences
of the adapter and two or three arbitrary
Nucleotides
• Analysis of the amplified fragments using gel
electrophoresis

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AFLPs: amplified fragment length
Polymorphisms
•A combination of PCR and RFLP
•Informative fingerprints of amplified
fragments

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AFLP-Major Steps
Amplified Fragment Length Polymorphism
• Genomic DNA double digests with a 4-cutter
(MseI) and a 6-cutter (EcoR1)
• Ligate adapters to the EcoR1 and MseI RE sites
• Primers complementary to Adapters with selective
nucleotides at 3’ ends and perform PCR
amplification
• Separate DNA fragments on high-resolution gels
• After detection, screen for band polymorphisms

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AFLP: Restriction and Ligation to Adapters

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AFLP: Pre-Selective Amplification
Primer (+ 1) for pre-selective amplification

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AFLP: Selective Amplification
Primer (+ 3) for selective amplification

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AFLP – band polymorphisms

30. Simple Sequence Repeats (SSRs)
Defined
• Simple sequence repeats (SSRs) or microsatellites
are tandemly repeated mono-,di-, tri-, tetra-, penta-,
and hexa-nucleotide motifs.
SSR length polymorphisms are caused by differences
in the number of repeats.
• SSR loci are “individually amplified by PCR using
pairs of oligonucleotide primers specific to unique DNA
sequences flanking the SSR sequence”.

31. Why Have SSRs Had Such a Large Impact
on Genomics?
• SSRs tend to be highly polymorphic.
• SSRs are highly abundant and randomly dispersed
throughout most genomes.
• Most SSR markers are co-dominant and locus
specific.
• Genotyping throughput is high and can be
automated.

32. SSR / microsatellite
1. Isolation of DNA fragments (vectors)
containing a simple sequence repeat
(microsatellite), e.g.
[AT]n [GC]n, [CGA]n, [GATA]n
1. Sequencing regions flanking the SSR
2. Designing primers for border sequences
3. Testing in population for duplications and
SSR polymorphism

33. SSR - methodolgy
Genotype A
Genotype A Genotype B
PCR amplification with
radiolabelled nucleoltide
Polyacrylamide Gel Electrophoresis
of PCR products and autoradiography
[AT]18 [AT]22
[AT]
18
[AT]
22
Genotype B

34. SSRs display multiple alleles

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39. Distribution of SNP
SINGLE NUCLEOTIDE POLYMORPHISM

40. Distribution of SNP
SNP

41. HUMAN SNP DISTRIBUTION
 Most common changes
Transitions:
Purines to Purines
Pyrimines to Pyrimidines
Transversion:
Purines to Pyrimidines
Pyrimidines to Purinces
 Single-base insertions & deletion
(indel)
Distribution of SNP

42. HOW SNP ARE INDICATED
Distribution of SNP

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55. Criteria for ideal DNA markers
• selectively neutral because they are usually located in non-coding regions of DNA
• High level of polymorphism
• unlimited in number and are not affected by environmental factors and/or the
developmental stage of the plant
• Even distribution across the whole genome (not clustered in certain regions)
• Co-dominance in expression (so that heterozygotes can be distinguished from
homozygotes)
• Clear distinct allelic features (so that the different alleles can be easily identified)
• Single copy and no pleiotropic effect
• Low cost to use (or cost-efficient marker development and genotyping)
• Easy assay/detection and automation
• High availability (un-restricted use) and suitability to be duplicated/multiplexed (so
that the data can be accumulated and shared between laboratories)
• No detrimental effect on phenotype
• However, no molecular markers fulfill all these characteristics. Researchers choose
the molecular marker according to their need and availability