GENETIC MARKERS

IN PLANT BREEDING
 Marker
 Gene of known function and location
 Gene that allows studying the inheritance of that gene
 Genetic information resides in the genome

Genetic Marker
Any phenotypic difference controlled by genes, that can be


used for studying recombination processes or selection of a

more or less closely associated target gene
Anything in the genome that is variable and can be used to
compare individuals
Detectable allelic variation on a chromosome can be a

phenotype, can also be a unique detectable sequence of DNA

 Genetic Marker
 Morphological marker
 Molecular marker
1. Protein marker
2. DNA marker
 Morphological Marker
•Phenotypic markers
•Naked eye marker

hulled naked Black white

 Molecular Markers

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

Types:
a) protein polymorphisms
b) DNA polymorphisms
 Molecular markers

Sequencing (SNPs)
Resolution power

Microsatellites (SSRs)

Multi-locus fingerprints (RFLP)

AFLP
(Amplified Fragment Length Polymorphism)
RAPD
(random amplified polymorphic DNA)

allozymes (protein-electrophoresis)
 Proteins Markers
Allozymes:
Isoenzymes of protein nature whose
synthesis is usually controlled by
codominant alleles and inherited by
monogenic ratios

Isozymes:
A species of enzyme that
exists into two or more
structural forms which are
easily identified by their
activities
 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 M2
Gene A Gene B
MFG

MFG
AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAAAAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG

Readily detectable sequence of DNA whose inheritance can be

monitored and associated with the trait inheritance
 Types of traits =types of markers

Single gene trait: seed shape Multigenic trait; ex: plant growth
=Quantitative Trait Loci

MFG MFG
 DNA Marker

1. Hybridization molecular based markers

2. PCR molecular based markers

Hybridization based markers

Examine differences in size of specific DNA restriction fragments

Require pure, high molecular weight DNA and probe
Usually performed on total cellular genome
Basis for DNA marker technology

• Restriction Endonucleases

• Polymerase chain reaction (PCR)

• DNA-DNA hybridization

•DNA sequencing
 RFLP based markers

*Examine differences in size of specific DNA

restriction fragments

*Require pure, high molecular weight DNA

*Usually performed on total cellular genome

 Endonucleases and restriction sequences
Name Taq I MboI Alu I Dde I Rsa I Scrf I Msp I Hae Ssp I
of the III
enzyme
Number 639 623 341 309 286 239 214 196 137
of
cutting
sites

Cutting
sites
TCGA GATC AG CT C TNAG GT AC CC NGG CC GG GG AAT ATT
CC

Note: N represent any base : A, T, C or G

YFG
AAATCGGGACCTAATGGGCC ATTTAGGGCAATTCCAAGGA

Ind 1 Ind 2
RFLP techniques
RFLP Polymorphisms interpretation

MFG
1 1 2 3 4 5 6

6
Advantages and disadvantages of RFLP

• Advantages • Disadvantages
– Reproducible – Time consuming
– Co-dominant – Expensive
– Simple – Use of radioactive
probes
 DNA/DNA Hybridization

Denaturation

Elevated temperature

Known DNA sequence

 Advantages and disadvantages

• Advantages • Disadvantages
– Reproducible – Time consuming
– Co-dominant – Expensive
– Simple – Use of radioactive
probes
 Polymerase Chain Reaction

Powerful technique for amplifying DNA

•

• Amplified DNA are then

separated by gel
electrophoresis
 PCR based
methods

1. Reactions conditions
*Target DNA ( or template)
*Reaction buffer containing the co-factor MgCl2
*One or more primers
*Four nucleotides (dATP, dCTP, dGTP, dTTP)
*Thermostable DNA polymerase
2. Use of DNA polymerase

= an enzyme that can synthesize DNA at

elevated temperature
ex : Taq = enzyme purified from hot
spring bacterium : Thermus aquaticus
3. Thermal cycle
*Denaturing step - one to several min at 94-96 º C
*Annealing step - one to several min at 50-65 º C
*Elongation step - one to several min at 72 º C

4. Repetition
–typically 20 to 50 times average 35 times
 PCR Based markers

Sequencing (SNPs)
Microsatellites (SSR)
AFLP (Amplified Fragment Length
Polymorphism)
RAPD (random amplified polymorphic
DNA)
 RAPD Markers
 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
 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
 RAPD

• Amplifies anonymous
stretches of DNA using
arbitrary primers
• Fast and easy method for
detecting polymorphisms

• Domimant markers
• Reproducibility
problems
 RAPD Markers
DNA markers which developed by amplifying random
sequence of specific markers through the used of random
primers
 RAPD Polymorphisms among landraces of sorghum

Sequences of 10-mer Name Sequence

RAPD primers

OP A08 5’ –GTGACGTAGG- 3’
OP
M A15 5’ –TTCCGAACCC- 3’
OP A 17 5’ –GACCGCTTGT- 3’
OP A19 5’ –CAAACGTCGG- 3’
RAPD gel configuration OP D02 5’ –GGACCCAACC- 3’
 RAPD
Advantages:
 Amplifies anonymous stretches of DNA using arbitrary
primers
 Fast and easy method for detecting polymorphisms

Disadvantages:
 Dominant markers

 Reproducibility problems
 RAPD Markers
 RAPD markers need to be converted to stable
PCR markers.
 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
 RAPD Polymorphisms among landraces of sorghum

Sequences of 10- Name Sequence

mer OP A08 5’ –GTGACGTAGG- 3’
RAPD primers OP A15 5’ –TTCCGAACCC- 3’
OP A 17
M 5’ –GACCGCTTGT- 3’
OP A19 5’ –CAAACGTCGG- 3’
OP D02 5’ –GGACCCAACC- 3’
RAPD gel configuration
 AFLP Markers
 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
 AFLP Markers
 The PCR products are then separated on
acrylamide gel
 128 linker combinations are readily
available
 Therefore 128 subsets can be amplified
 Patented technology
 AFLP Markers
 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

 SSR repeats and primers

Repeat
Sequence

GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC
GGT(5)
GAGGGCTGATGAGGTGGATA
ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGAT
TCCGGTCGTCAGAATCAGAATCAGAATCGATATGGTG
GCAGTGGTGGTGGTGGTGGTGGTTTTGGTGGTGGTGA
ATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTGGT
AAGAAACCTCTTCCTGTTAG
ATCTTATGGCGGTTCTCGTG
ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCT
GCTGTGGAACCA…..
 SSR (Simple sequence repeat)
DNA markers which developed by amplifying microsatellite in
the genome

Sequence Primer
ACTGTCGACACACACACACACGCTAGCT (AC)7
TGACAGCTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACGCTAGCT
(AC)8
TGACAGCTGTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACACACGCTAGCT
(AC)10
TGACAGCTGTGTGTGTGTGTGTGTGTGCGATCGA

ACTGTCGACACACACACACACACACACACACGCTAGCT
(AC)12
TGACAGCTGTGTGTGTGTGTGTGTGTGTGTGCGATCGA
 SSR polymorphisms

P1 AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG

P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG

P1 P2

Gel configuration
Linkage groups
SSR scoring for F 5:6 pop from the cross
Anand x N97-3708-13

N97
Anand

M
 SNPs
(Single Nucleotide Polymorphisms)
DNA markers which their polymorphism can be determined by single
nucleotide difference
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.

 DNA sequencing

Sequencer

Sequencing gel Sequencing graph

 Genetic marker characteristics
Characteristic Morphologica Protein RFLP RAPD SSR markers
s l markers markers markers markers
Number of Limited Limited Almost Unlimited High
loci unlimited
Inheritance Dominant Codominant Codominant Dominant Codominant
Positive Visible Easy to detect Utilized Quick assays Well
features before the with many distributed
latest markers within the
technologies genome,
were available many
polymorphism
Negative Possibly Possibly Radioactivity High basic Long
features negative tissue specific requirements, investment development
linkage to rather of the
other expensive markers,
characters expensive
Co-dominant marker
Gel configuration Polymorphism
P1 P2 O1 O2 -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 : ????
 Desirable properties
 Polymorphic
 Co-dominant inheritance
 Occurs throughout the genome
 Reproducible
 Easy, fast and cheap to detect
 Selectivity neutral
 High resolution with large number of samples
 Eukaryotic Genes and Genomes
genome = DNA content of a complete haploid set of
hromosomes= DNA content of a gamete (sperm
or egg) DNA year
Chromosom content/ sequence genes/
Species cM
es haploid(M complete haploid
b) d
1 N/A 5 1997 4,200
E. coli
16 4000 12 1997 5,800
S. cerevisiae

6 300 100 1998 19,000

C. elegans

D. 4 280 180 2000 14,000

melanogaster
2002 draft
M. musculus 20 1700 3000 30,000?
2005 finished?
2001 draft
H. sapiens 23 3300 3000 30,000?
2003 finished
Note: cM = centi Morgan = 1% recombination
Mb = megabase = 1 million base-pairs of DNA
Kb = kilobase = 1 thousand base-pairs of DNA
 DNA true
generation design
Species cM content/ breeding
time crosses?
haploid (Mb) strains?

E. coli N/A 5 30 min yes yes

S. cerevisiae 4000 12 90 min yes yes

C. elegans 300 100 4d yes yes

D. melanogaster 280 180 2 wk yes yes

M. musculus 1700 3000 3 mo yes yes

H. sapiens 3300 3000 20 yr no no

•Human genetics is (vs prospective). Human geneticists
retrospective cannot test hypotheses prospectively.
The mouse provides a prospective
surrogate.
• Can’t do selections

• Meager amounts of data Human geneticists typically rely upon statistical

arguments as opposed to overwhelming
amounts of data in drawing connections
between genotype and phenotype.

• Highly dependent on DNA-based maps and DNA-based analysis

The unique advantages of human genetics:

• A large population which is self-screening to a considerable degree

• Phenotypic subtlety is not lost on the observer
• The self interest of our species
A locus is said to be polymorphic if two or more alleles are each present at
a frequency of at least 1% in a
population
of animals.

1) SNPs = single nucleotide polymorphisms = single nucleotide

substitutions

In human
populations:

Hnuc = average heterozygosity per nucleotide =

site 0.001
 SYNONOMOUS CHANGES

TTT GCT GGC CAC TTT GCT GGA CAC

Phe Ala Gly His Phe Ala Gly His

NON-SYNONOMOUS CHANGES

TTT GCT GGC CAC TTT GCT TGC CAC

Phe Ala Gly His Phe Ala Cys His

The great majority (probably 99%) of SNPs are selectively “neutral” changes
of little or no functional consequence:

•outside coding or gene regulatory regions (>97% of human

genome)

• silent substitutions in coding sequences

• some amino acid substitutions do not affect protein stability or

function

•disadvantageous SNPs selected against --> further

underrepresentation

A small minority of SNPs are of functional consequence and are

selectively advantageous or disadvantageous.
 molecular 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 M2
Gene A Gene B
MFG
MFG
AACCTGAAAAGTTACCCTTTAAAGGCTTAAGGAAAAAGGGTTTAACCAAGGAATTCCATCGGGAATTCCG

readily detectable sequence of DNA whose inheritance can be

monitored and associated with the trait inheritance
Image from UV light Image from computer
table screen
Dominant versus Co-dominant

Dominant:
No distinction between homo- and heterozygotes
possible

No allele frequencies available

AFLP, RAPD

Co-dominant:
homozygotes can be distinguished from
heterozygotes; allele frequencies can be calculated

microsatellites, SNP, RFLPs

Desirable properties for a good
molecular marker

* Polymorphic

* Co-dominant inheritance

* Occurs throughout the genome

* Reproducible

* Easy, fast and cheap to detect

* Selectivity neutral

* High resolution with large number of

samples