Transcript THE USE OF GENETIC MARKERS IN PLANT BREEDING Use of Molecular Markers  Clonal identity,  Family structure,  Population structure,  Phylogeny (Genetic Diversity)  Mapping 

THE USE OF
GENETIC MARKERS
IN PLANT BREEDING
Use of Molecular Markers
 Clonal identity,
 Family structure,
 Population structure,
 Phylogeny (Genetic Diversity)
 Mapping
 Parental analysis,
 Gene flow,
 Hybridisation
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
Clonal Identity
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
Genetic 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 (Quantitative Trait Loci)

A locus or DNA segment that carries more genes coding for an
agronomic or other traits
 Individual loci responsible for quantitative genetic variation
 Region in the genome containing factors influencing a
quantitative trait
 Region identified by statistical association
QTL Mapping


A set of procedures for detecting genes controlling quantitative traits (QTL) and
estimating their genetics effects and location
Localizing and determining a segment of DNA that regulate quantitative traits
 Detecting and locating gene having an effect on a quantitative traits
To assist selection
Marker Assisted Selection

Types of traits
Single gene trait: seed shape
Multigenic trait; ex: plant growth
=Quantitative Trait Loci
Linkage groups
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
Conduct linkage analysis to develop QTL Marker
Other methods to develop population for markers exist
but are more expensive and slower to develop
→ Near Isogenic Lines, Recombinant Inbreeds, Single
Seed Decent
MAS

Marker assisted selection
The use of DNA markers that are tightly-linked

to target loci as a substitute for or to assist
phenotypic screening
Assumption
DNA markers can reliably predict phenotype
Marker Assisted Selection
 Breeding for specific traits in plants 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.
CONVENTIONAL PLANT BREEDING
P1
x
P2
Donor
Recipient
F1
large populations consisting of thousands of plants
F2
PHENOTYPIC SELECTION
Salinity screening in
phytotron
Glasshouse trials
Bacterial blight screening
Phosphorus deficiency plot
Field trials
MARKER-ASSISTED BREEDING
P1
x
P2
Resistant
Susceptible
F1
F2
large populations consisting of thousands of plants
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers
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 in rice

Increased reliability
• No environmental effects
• Can discriminate between homozygotes and
heterozygotes and select single plants
Potential benefits from MAS

more accurate and
efficient selection of
specific genotypes
• May lead to accelerated
variety development

Crossing house
more efficient use of
resources
• Especially field trials
Backcross nursery
Overview of
‘marker
genotyping’
(1) LEAF TISSUE
SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS
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
Conduct linkage analysis to develop QTL Marker
Other methods to develop population for markers exist
but are more expensive and slower to develop
→ Near Isogenic Lines, Recombinant Inbreeds, Single
Seed Decent
Considerations for using DNA
markers in plant breeding

Technical methodology
• simple or complicated?





Reliability
Degree of polymorphism
DNA quality and quantity required
Cost**
Available resources
• Equipment, technical expertise
Markers must be
tightly-linked to target loci!

Ideally markers should be <5 cM from a gene or QTL
RELIABILITY FOR
SELECTION
Marker A
5 cM
Using marker A only:
QTL
1 – rA = ~95%
Marker B
Marker A
5 cM
QTL
Using markers A and B:
5 cM
1 - 2 rArB = ~99.5%
• Using a pair of flanking markers can greatly improve reliability
but increases time and cost
Markers must be polymorphic
RM84
1 2
7 8
3 4 5 6
RM296
1
8
2
3
4 5
P1 P2
P1 P2
Not polymorphic
Polymorphic!
6
7
DNA extractions
Mortar and pestles
Porcelain grinding plates
LEAF SAMPLING
Wheat seedling tissue sampling
in Southern Queensland,
Australia.
High throughput DNA extractions “GenoGrinder”
PCR-based DNA markers


Generated by using Polymerase Chain Reaction
Preferred markers due to technical simplicity and cost
PCR Buffer +
MgCl2 +
dNTPS +
PCR
Taq +
Primers +
DNA template
THERMAL CYCLING
GEL ELECTROPHORESIS
Agarose or Acrylamide gels
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
BREEDING SCHEMES
1.
2.
3.
4.
Marker-assisted backcrossing
Pyramiding
Early generation selection
‘Combined’ approaches
Marker-assisted backcrossing
(MAB)

MAB has several advantages over conventional
backcrossing:
• Effective selection of target loci
• Minimize linkage drag
• Accelerated recovery of recurrent parent
1
2
3
4
1
2
3
4
1
2
3
4
Target
locus
TARGET LOCUS
SELECTION
FOREGROUND
SELECTION
RECOMBINANT
SELECTION
BACKGROUND
SELECTION
BACKGROUND SELECTION
Gene Pyramiding
 Widely used for combining multiple disease resistance
genes for specific races of a pathogen
 Pyramiding is extremely difficult to achieve using
conventional methods
Consider: phenotyping a single plant for multiple forms of
seedling resistance – almost impossible
 Important to develop ‘durable’ disease resistance against
different races
Process of combining several genes, usually from 2 different parents,
together into a single genotype
Breeding plan
P1
x
P1
Gene A
Gene B
Genotypes
P1: AAbb
F1
Gene A + B
F2
MAS
Select F2 plants that have
Gene A and Gene B
P2: aaBB
x
F1: AaBb
F2
AB
Ab
aB
ab
AB
AABB
AABb
AaBB
AaBb
Ab
AABb
AAbb
AaBb
Aabb
aB
AaBB
AaBb
aaBB
aaBb
ab
AaBb
Aabb
aaBb
aabb
Early generation MAS


MAS conducted at F2 or F3 stage
Plants with desirable genes/QTLs are selected
and alleles can be ‘fixed’ in the homozygous
state
• plants with undesirable gene combinations can be
discarded

Advantage for later stages of breeding program
because resources can be used to focus on
fewer lines
P1
x
Susceptible
P2
Resistant
F1
F2
large populations (e.g. 2000 plants)
MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes
MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes
SINGLE-LARGE SCALE MARKERASSISTED SELECTION (SLS-MAS)
PEDIGREE METHOD
P1
x
P2
P1
F1
F2
x
P2
F1
Phenotypic
screening
Plants spaceplanted in rows
for individual
plant selection
F3
F3
Families grown
in progeny rows
for selection.
F4
F5
Families grown
in progeny rows
for selection.
F5
Pedigree
selection based
on local needs
F6
F6
F7
Further
yield trials
F7
Multi-location testing, licensing, seed
Only desirable
F3 lines planted
in field
F4
Preliminary yield
trials. Select
single plants.
F8 – F12 increase and cultivar release
MAS
F2
Multi-location testing, licensing, seed
F8 – F12 increase and cultivar release
Benefits: breeding program can be efficiently
scaled down to focus on fewer lines
Combined approaches

In some cases, a combination of phenotypic
screening and MAS approach may be useful
1. To maximize genetic gain (when some QTLs have
been unidentified from QTL mapping)
2. Level of recombination between marker and QTL
(in other words marker is not 100% accurate)
3. To reduce population sizes for traits where marker
genotyping is cheaper or easier than phenotypic
screening
‘Marker-directed’ phenotyping
(Also called ‘tandem selection’)
P1 (S) x P2 (R)
Recurrent
Parent
Donor
Parent
F1 (R) x P1 (S)
BC1F1 phenotypes: R and S
Use when markers are not
100% accurate or when
phenotypic screening is
more expensive compared
to marker genotyping
MARKER-ASSISTED SELECTION (MAS)
1 2
3
4
5 6 7
8
9 10 11 12 13 14 15 16 17 18 19 20 …
SAVE TIME & REDUCE
COSTS
PHENOTYPIC SELECTION
*Especially for quality traits*