GENETIC MARKERS
IN PLANT BREEDING
Marker
Gene of known function and location, or a
mutation within a gene that allows studying the
inheritance of that gene
Genetic information resides in the genome
Genetic Marker
Any phenotypic difference controlled by the genes, that
can be used for studying recombination processes or
selection of a more or less closely associated target gene
Genetic Marker
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Morphological marker
Molecular marker
Readily detectable sequence of protein or DNA that are closely
linked to a gene locus and/or a morphological or other characters
of a plant
Readily detectable sequence of protein or DNA whose inheritance
can be monitored and associated with the trait inheritance
independently from the environment
1. Protein marker
2. DNA marker
Molecular markers
Sequencing (SNPs)
Microsatellites (SSRs)
AFLP
(Amplified Fragment Length Polymorphism)
RAPD
(random amplified polymorphic DNA)
chloroplastDNA PCR-RFLP
allozymes (protein-electrophoresis)
Morphological marker
(phenotypic/naked eye marker)
2-rowed
6-rowed
hulled
naked
Black
white
non-waxy
waxy
Karl Von Linne (1707-1778)
Molecular markers
Important aspect:
 Polymorphism
The existence of two or more forms that are
genetically distinct from one another but contained
within the same interbreeding population
 Pattern of inheritance
The pattern of genetic information transmission
from parents to progeny
Polymorphism
Co-dominant marker
Gel configuration
P1
P2
O1
O2
Polymorphism
-Parent 1 : one band
-Parent 2 : a smaller band
-Offspring 1 : heterozygote = both
bands
-Offspring 2 : homozygote parent 1
Dominant marker
Polymorphism
Gel configuration
Parent 1 : one band
P1
P2
O1
O2
-Parent 2 : no band
-Offspring 1 : homozygote parent 1
-Offspring 2 : ????
Dominant versus Co-dominant
Dominant
No distinction between homo- and heterozygotes possible
No allele frequencies available
RAPD
Co-dominant
Homozygotes can be distinguished from heterozygotes;
Allele frequencies can be calculated
microsatellites, SNP, RFLPs
Desirable properties for a good
molecular marker
 High Polymorphic
Co-dominant inheritance
Occurs throughout the genome
Reproducible
Easy, fast and cheap to detect
Selectivity neutral
High resolution with large number of samples
Nondestructive assay
Random distribution throughout the genome
Assay can be automated
Protein markers
Genetic markers which based on protein
polymorphisms
a. Allozyme
isoenzymes of proteins nature whose synthesis is usually
controlled by codominant alleles and inherited by monogenic
ratios. They show a specific banding pattern if separated by
electrophoresis
b. Isozyme
A species of enzyme that exists in two or more structural
form, which are easily identified by electrophoretic methods
Proteins Polymorphisms
Seed storage proteins
Isozymes
Isozyme
Isozyme
Starch gel of the isozyme malate dehydrogenase (MDH). The numbers
indicate first the MDH locus, and next the allele present (ie. 3-18 is locus 3
allele 18). Some bands are heterodimers (intralocus or interlocus).
DNA marker
Segments of DNA with an identifiable physical
location on a chromosome and whose inheritance can
be followed
A marker can be a gene, or it can be some section of DNA with
no known function
Types of DNA Marker can be differentiated based on
molecular technique used to develop the marker
1. Restriction enzymes
2. Hybridization
3. PCR
4. Sequencing
DNA structure
Chromosome to DNA
Stretch of nitrogen fixation gene in soybean
1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag
gcgggcgtcg ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc
cattcgcctg 181 ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc
cagagtgctt gatgccatgc tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc
agccatggaa cgtgctcgcc 361 agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421
ctgagtccaa caacatagca attaaggtag gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc
cagaagcaaa aaagagggaa cctgacacta 541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat
601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt ggaggacagc tttgtggagt 661 ctgaaatcac catctacctt
tacttaggtt ctgagtgcca aacccaaggc accaggcatg 721 cgtccttgac tccggagcca tcaggcaggc tttcctcagc cttttgcagc
caagtctttt 781 agcctattgg tctgagttca gtgtggcagt tggttaggaa agaaggtggt tcttcgacca 841 ctaacagttt
ggatttttta ggatgctagt cctttaaaa ……….
DNA marker
1 ccacgcgtcc gtgaggactt gcaagcgccg cggatggtgg gctctgtggc tgggaacatg 61 ctgctgcgag ccgcttggag gcgggcgtcg
ttggcggcta cctccttggc cctgggaagg 121 tcctcggtgc ccacccgggg actgcgcctg cgcgtgtaga tcatggcccc cattcgcctg 181
ttcactcaga ggcagaggca gtgctgcgac ctctctacat ggacgtacag gccaccactc 241 ctctggatcc cagagtgctt gatgccatgc
tcccatacct tgtcaactac tatgggaacc 301 ctcattctcg gactcatgca tatggctggg agagcgaggc agccatggaa cgtgctcgcc 361
agcaagtagc atctctgatt ggagctgatc ctcgggagat cattttcact agtggagcta 421 ctgagtccaa caacatagca attaaggtag
gaggagggat ggggatgttg tgtggccgac 481 agttgtgagg ggttgtggga agatggaagc cagaagcaaa aaagagggaa cctgacacta
541 tttctggctt cttgggttta gcgattagtg cccctctctc atttgaactc aactacccat 601 gtctccctag ttctttctct gcctttaaaa aaaaatgtgt
ggaggacagc tttgtggag
DNA
M1
Gene A
M2
MFG
Gene B
MFG
AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAAAAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG
readily detectable sequence of DNA whose inheritance can be
monitored and associated with the trait inheritance
Image from UV light table
Image from computer screen
Basis for DNA marker
technology
•Restriction Endonucleases
•DNA-DNA hybridization
•Polymerase chain reaction (PCR)
•DNA sequencing
RFLP techniques
RFLP Polymorphisms interpretation
MFG
1
2
3
4
5
6
1
2
3
4
5
6
RFLP based markers
Examine differences in size of specific DNA restriction fragments
Require pure, high molecular weight DNA
Usually performed on total cellular genome
Advantages and disadvantages of RFLP
• Advantages
– Reproducible
– Co-dominant
– Simple
• Disadvantages
– Time consuming
– Expensive
– Use of probes
AFLP Markers
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Most complex of marker technologies
Involves cleavage of DNA with two different enzymes
Involves ligation of specific linker pairs to the digested
DNA
Subsets of the DNA are then amplified by PCR
The PCR products are then separated on acrylamide gel
128 linker combinations are readily available
Therefore 128 subsets can be amplified
AFLP Markers
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Technically demanding
Reliable and stable
Moderate cost
Need to use different kits adapted to the size of
the genome being analyzed.
Like RAPD markers need to be converted to
quick and easy PCR based marker
RAPD
 Amplifies anonymous stretches
of DNA using arbitrary primers
 Fast and easy method for
detecting polymorphisms
• Domimant markers
• Reproducibility problems
RAPD Polymorphisms
among landraces of sorghum
Sequences of 10-mer
RAPD primers
RAPD gel configuration
Name
Sequence
OP A08
M
OP A15
OP A 17
OP A19
OP D02
5’ –GTGACGTAGG- 3’
5’ –TTCCGAACCC- 3’
5’ –GACCGCTTGT- 3’
5’ –CAAACGTCGG- 3’
5’ –GGACCCAACC- 3’
RAPD Markers
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There are other problems with RAPD markers
associated with reliability
Because small changes in any variable can
change the result, they are unstable as markers
RAPD markers need to be converted to stable
PCR markers.
How?
RAPD Markers
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The polymorphic RAPD marker band is isolated
from the gel
It is used a template and re-PCRed
The new PCR product is cloned and sequenced
Once the sequence is determined, new longer
and specific primers can be designed
VNTR
Variable Number of Tandem Repeats
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Tandem repeats (TR):
DNA sequences which are existed in repeated numbers in
the genome
• Satellite DNA
• Minisatellites
• Microsatellites
Variable Number (VN)
High polymorphism in number of repeats
VNTR
Variable Number of Tandem Repeats
• Satellite DNA
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2-250 bp repeat unit size
Constitutes 1- 60% of the genome
Some can be separated in CsCl
• ‘satellite band’
• Minisatellites
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9-50 bp repeat unit size
100 – 1000 x repeated
• Microsatellites
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2-6 bp repeat unit size
10s – 100 x repeated
Microsatellites
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Short tandem repeats (simple sequence repeat)
• 2 – dinucleotides
• 3 – trinucleotides
• 4 – tetranucleotides
Randomly distributed in genome
Non-coding
• Some within coding sequences
 Especially trinucleotides
• Some related to diseases
Nomenclature
• Perfect
GCTAGCCACACACACACACATGCATC
• Interrupted
GCTAGCCACACGTCACACACTGCATC
• Compound
GCTAGCCACACATATATGTGTGCATC
SSR repeats and primers
Repeat
GGT(5)
Sequence
GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC
ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCC
GGTCGTCAGAATCAGAATCAGAATCGATATGGTGGCAGTGG
TGGTGGTGGTGGTGGTTTTGGTGGTGGTGAATCTAAGGCG
GATGGAGTGGATAATTGGGCGGTTGGTAAGAAACCTCTTCC
TGTTAG
ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCT
GTGGAACCA…..
SSR polymorphisms
P1
AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG
P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG
P1
Gel configuration
P2
SNP
(Single Nucleotide Polymorphisms)
SNPs on a DNA strand
Hybridization using fluorescent dyes
• Any two unrelated individuals differ by one base pair every 1,000
or so, referred to as SNPs.
• Many SNPs have no effect on cell function and therefore can be
used as molecular markers.
Genetic marker characteristics
Characteristics
Morphological
markers
Protein
markers
RFLP markers
RAPD
markers
Number of
loci
Limited
Limited
Almost
unlimited
Unlimited
High
Inheritance
Dominant
Codominant
Codominant
Dominant
Codominant
Positive
features
Visible
Easy to detect
Utilized before Quick assays
the latest
with many
technologies
markers
were available
Well
distributed
within the
genome, many
polymorphism
Negative
features
Possibly
negative
linkage to
other
characters
Possibly tissue
specific
Radioactivity
requirements,
rather
expensive
Long
development
of the
markers,
expensive
High basic
investment
SSR markers
Developing a Marker
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Best marker is DNA sequence responsible for
phenotype i.e. gene
If you know the gene responsible and has been
isolated, compare sequence of wild-type and
mutant DNA
Develop specific primers to gene that will
distinguish the two forms
Developing a Marker
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If gene is unknown, screen contrasting
populations
Use populations rather than individuals
Need to “blend” genetic differences between
individual other than trait of interest
Developing Markers
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Cross individual differing in trait you wish to develop a
marker
Collect progeny and self or polycross the progeny
Collect and select the F2 generation for the trait you are
interested in
Select 5 - 10 individuals in the F2 showing each trait
Extract DNA from selected F2s
Pool equal amounts of DNA from each individual into
two samples - one for each trait
Screen pooled or “bulked” DNA with what method of
marker method you wish to use
Types of traits
(types of markers)
Single gene trait: seed shape
MF
G
Multigenic trait; ex: plant growth
=Quantitative Trait Loci
MFG
USES OF MOLECULAR MARKER
 Clonal identity
 Parental analysis
 Family structure
 Population structure
 Gene flow
 Hybridisation
 Phylogeny
 Measure genetic diversity
 Mapping
 Tagging
Genetic Diversity
 Define appropriate geographical scales for monitoring
and management (epidemology)
 Establish gene flow mechanism
 identify the origin of individual (mutation detection)
 Monitor the effect of management practices
 manage small number of individual in ex situ collection
 Establish of identity in cultivar and clones (fingerprint)
 paternity analysis and forensic
Genetic Diversity
Mapping
The determination of the position and relative distances
of gene on chromosome by means of their linkage
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Genetic map
A linear arrangement of genes or genetic markers obtained based on
recombination
An ordering of genes and markers in a linear arrangement
corresponding to their physical order along the chromosome,
based on linkage.
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Physical map
A linear order of genes or DNA fragments
An ordering of landmarks on DNA, regardless of inheritance,
measured in base pairs.
Physical Mapping
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It contains ordered overlapping cloned DNA fragment
The cloned DNA fragments are usually obtained using
restriction enzyme digestion
QTL Mapping
A set of procedures for detecting genes controlling
quantitative traits (QTL) and estimating their
genetics effects and location
 To assist selection
Fundamental Genetics
(Background for Linkage Analysis)
 Rule of Segregation
• offspring receive ONE allele (genetic material)
from the pair of alleles possessed by BOTH
parents
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Rule of Independent Assortment
• alleles of one gene can segregate independently of
alleles of other genes
• (Linkage Analysis relies on the violation of
Independent Assortment Rule)
Linkage Analysis
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Goal:
find a marker “linked” to a disease gene.
LOD score = log of likelihood ratio
LR[θ;data] == k P[data; θ]
θ = estimate of genetic distance
(recombination fraction) between marker and
quantitative traits
= proportion of recombinant gametes/total gametes
Linkage Analysis
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Genes near each other on a chromosome tend
to be inherited together, that is, they are linked.
Linkage analysis are the techniques used to
identify such linkages among genes
Linkage groups which include genetic markers
and genes determinative of phenotype allow the
identification of determinative alleles (and
therefore prediction)
Linkage
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Mendel showed that alleles segregate
independently. Then he tested genes
Sometimes inheritance of two genes are
independent of another, that is phenotype ratios
are 9:3:3:1
Sometimes inheritance of two genes are linked
together, showing a ratio of 3:0:0:1
Linkage can vary continuously from perfectly
correlated to uncorrelated.
Why genes are linked
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Alleles are arranged linearly
Each parent passes only one of its two chromosomes
to an offspring.
Recombination periodically switches which
chromosome in the parent is passed along
Alleles near each other are more likely to be passed
along than ones further apart
Alleles on different chromosomes are always inherited
independently.
Marker Assisted Selection
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Breeding for specific traits in plants and animals
is expensive and time consuming
The progeny often need to reach maturity
before a determination of the success of the
cross can be made
The greater the complexity of the trait, the more
time and effort needed to achieve a desirable
result.
MAS
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The goal to MAS is to reduce the time needed to
determine if the progeny have trait
The second goal is to reduce costs associated
with screening for traits
If you can detect the distinguishing trait at the
DNA level you can identify positive selection
very early.
Marker Assisted Breeding
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MAS allows for gene pyramiding - incorporation
of multiple genes for a trait
Prevents development of biological resistance to
a gene
 Reduces space requirements - dispose of
unwanted plants and animal early
QTL study
Trait M. 1 M. 2 M. 3
P.1
P.2
I.1
I.2
I.3
I.4
2.5
8.4
7.1
2.5
4.5
2.3
1
3
3
2
2
1
1
3
1
1
3
1
1
3
1
1
2
3
Statistical programs used in molecular marker studies
* SAS
* ANOVA
* Mapmaker
* Cartographer
Types of population used for molecular markers studies:
F2, RILs, Backcrosses (MILs), DH.
QTL Mapping
Recombination picture
Crossover is the alternation of allele generating chromatid
(half of chromosome)