Transcript Slide 1

GENETIC MARKERS
IN PLANT BREEDING
MARKERS IN BIOLOGY
1. Phenotypic markers
= Naked eye markers
Flower colors, shape of pods, etc..
P = E+G
Karl Von Linne (1707-1778)
2. Genotypic (molecular) markers
Readily detectable sequence of protein or
DNA whose inheritance can be monitored and
associated with the trait inheritance
independently from the environment:
a) protein polymorphisms
b) DNA polymorphisms
Molecular markers
Sequencing (SNPs)
Microsatellites (SSRs)
Multi-locus fingerprints
AFLP
(Amplified Fragment Length Polymorphism)
RAPD
(random amplified polymorphic DNA)
chloroplastDNA PCR-RFLP
allozymes (protein-electrophoresis)
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).
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
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
Molecular 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
Usually performed on total cellular genome
DNA/DNA Hybridization
Denaturation
Elevated temperature
Restriction
Fragment Length
Polymorphism
Known DNA sequence
Endonucleases and restriction sequences
Name
of the
enzyme
Number
of
cutting
sites
Taq I
MboI
Alu I
Dde I
Rsa I
Scrf I
Msp I
Hae
III
Ssp I
639
623
341
309
286
239
214
196
137
AG CT
C TNAG
GT AC
CC NGG
CC GG
GG
CC
AAT ATT
Cutting
sites
TCGA
GATC
Note: N represent any base : A, T, C or G
AAATCGGGACCTAATGGGCC
Ind 1
YFG
ATTTAGGGCAATTCCAAGGA
Ind 2
RFLP techniques
RFLP Polymorphisms interpretation
MFG
1
2
3
4
5
6
1
2
3
4
5
6
Advantages and disadvantages of
RFLP
• Advantages
– Reproducible
– Co-dominant
– Simple
• Disadvantages
– Time consuming
– Expensive
– 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
Example:
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
Molecular markers which developed by amplifying random sequence
of specific markers through the used of random primers
 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
 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
Disadvantages:
 Domimant markers
 Reproducibility problems
RAPD Polymorphisms among landraces of sorghum
Sequences of 10-mer
RAPD primers
RAPD gel configuration
Name
Sequence
OP A08
OP A15
M
OP A 17
OP A19
OP D02
5’ –GTGACGTAGG- 3’
5’ –TTCCGAACCC- 3’
5’ –GACCGCTTGT- 3’
5’ –CAAACGTCGG- 3’
5’ –GGACCCAACC- 3’
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
 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
Molecular markers which developed by amplifying microsatellite in
the genome
Repeat
GGT(5)
Sequence
GCGCCGAGTTCTAGGGTTTCGGAATTTGAACCGTC
GAGGGCTGATGAGGTGGATA
ATTGGGCGTCGGTGAAGAAGTCGCTTCCGTCGTTTGATTCCGGTCGTCA
GAATCAGAATCAGAATCGATATGGTGGCAGTGGTGGTGGTGGTGGTGGT
TTTGGTGGTGGTGAATCTAAGGCGGATGGAGTGGATAATTGGGCGGTTG
GTAAGAAACCTCTTCCTGTTAG
ATCTTATGGCGGTTCTCGTG
ATTCTGGAATGGAACCAGATCGCTGGTCTAGAGGTTCTGCTGTGGAACC
A…..
SSR polymorphisms
P1
AATCCGGACTAGCTTCTTCTTCTTCTTCTTTAGCGAATTAGG
P2 AAGGTTATTTCTTCTTCTTCTTCTTCTTCTTCTTAGGCTAGGCG
P1
Gel configuration
P2
SSR scoring for F 5:6 pop from the cross
Anand x N97-3708-13
M
SNPs
(Single Nucleotide Polymorphisms)
Molecular 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
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
Co-dominant marker
Gel configuration
P1
P2
O1
O2
Dominant marker
P2
O1
Polymorphism
Parent 1 : one band
Gel configuration
P1
Polymorphism
-Parent 1 : one band
-Parent 2 : a smaller band
-Offspring 1 : heterozygote =
both bands
-Offspring 2 : homozygote
parent 1
O2
-Parent 2 : no band
-Offspring 1 : homozygote parent 1
-Offspring 2 : ????
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
Use of Molecular Markers
Clonal identity
Parental analysis
Family structure
Population structure
Gene flow
Phylogeography
Hybridisation
Phylogeny
USES OF MOLECULAR MARKER

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
Gotcha!
fingerprints
seeds,
plantlets
early selection
of the good allele
Mapping
The determination of the position and relative
distances of gene on chromosome by means of
their linkage

Genetic map
A linear arrangement of genes or genetic markers obtained based on
recombination

Physical map
A linear order of genes or DNA fragments
Physical Mapping
It contains ordered overlapping cloned DNA
fragment
The cloned DNA fragments are usually obtained
using restriction enzyme digestion


Molecular Maps
Molecular markers (especially RFLPs and SSRs) can be used to
produce genetic maps because they represent an almost
unlimited number of alleles that can be followed in progeny of
crosses.
Chromosomes with
morphological
marker alleles
Chromosomes with molecular
marker alleles
RFLP1b
RFLP2b
SSR1b
T
t
r
R
or
RFLP1a
RFLP2a
SSR1a
RFLP3b
RFLP3a
SSR2b
SSR2a
RFLP4b
RFLP4a
QTL Mapping
A set of procedures for detecting genes controlling
quantitative traits (QTL) and estimating their
genetics effects and location
 To assist selection
Types of traits
Single gene trait: seed shape
Multigenic trait; ex: plant growth
=Quantitative Trait Loci
MFG
MFG
Making A Linkage Map
R642
RZ141
G320
G44
RG2
C189
G1465
Rice chromosome 11
Genotype
G320 RG2 C189
A
A
A
A
A
B
A
B
A
A
B
B
B
A
A
B
B
A
B
A
B
B
B
B
Total
No. of
Individuals
47
8
5
15
19
24
3
42
.
163
Recombinants between G320 and RG2 = 5 + 15 + 19 + 3 = 42 = 26%
Recombinants between RG2 and C189 = 8 + 5 + 24 + 3 = 40 = 25%
Recombinants between G320 and C189 = 8 + 15 + 19 + 24 = 66 = 40%
Making a Linkage Map
A
A
A
G320 RG2
C189
A
A
A
B
B
A
Frequency of Genotype
B
B
A
47
8
5
15
19
24
3
42
Making a Lingkage Map
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
Linkage groups
Marker Assisted Selection
 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
 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 Selection
Useful when the gene(s) of interest is difficult to select
1. Recessive Genes
2. Multiple Genes for Disease Resistance
3. Quantitative traits
4. Large genotype x environment interaction
Marker Assisted Selection
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
Developing a Marker



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



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




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
Developing Markers




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
Method is called “Bulked Segregant
Analysis”
Marker Development



Other methods to develop population
for markers exist but are more
expensive and slower to develop
Near Isogenic Lines, Recombinant
Inbreeds, Single Seed Decent
What is the advantage to markers in
breeding?