GENETIC MARKERS

IN PLANT BREEDING
 Use of Molecular Markers
 Clonal identity,
 Family structure,
 Phylogeny (Genetic Diversity)
 Mapping
 Population structure,
 Parental analysis
Clonal Identity

fingerprints

seeds,
plantlets
early selection
of the good allele
 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

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 Chromosomes with molecular

morphological marker alleles
marker alleles RFLP1b RFLP1a
RFLP2b RFLP2a
SSR1b SSR1a
RFLP3b RFLP3a
T t
SSR2b SSR2a

R r RFLP4b RFLP4a

or
 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

ÞTo assist selection

Marker Assisted Selection
 Types of traits
Multigenic trait; ex: plant
Single gene trait: seed shape
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

Recipient Donor
F1
large populations consisting of thousands
F2 of plants

PHENOTYPIC SELECTION

Salinity screening in
phytotron
Bacterial blight screening Phosphorus deficiency plot

Glasshouse trials Field trials

 MARKER-ASSISTED BREEDING

P1 x P2
Susceptible Resistant

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 Crossing house

development
• more efficient use of
resources
• Especially field trials

Backcross nursery
Overview of (1) LEAF TISSUE
‘marker SAMPLING
genotyping’
(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

QTL
Using marker A only:
5 cM
1 – rA = ~95%
Marker A Marker B
QTL Using markers A and B:
5 cM 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 RM296
1 2 3 4 5 6 1 2 3 4 5 6 7
7 8 8
P1 P2
P1 P2

Not polymorphic Polymorphic!

 DNA extractions

Mortar and pestles

Porcelain grinding plates

LEAF SAMPLING

Wheat seedling tissue sampling in

Southern Queensland, Australia.

High throughput DNA extractions

“Geno-Grinder”
 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
 Agarose gel electrophoresis

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

UV transilluminator

UV light
Acrylamide gel electrophoresis 1

UV transilluminator

UV light
Acrylamide gel electrophoresis 2
 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. Marker-assisted backcrossing
2. Pyramiding
3. Early generation selection
4. ‘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 RECOMBINANT BACKGROUND

SELECTION SELECTION SELECTION

FOREGROUND
BACKGROUND SELECTION
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 Genotypes

P1 x P1
P1: AAbb x P2: aaBB
Gene A Gene B

F1
F1: AaBb
Gene A + B

F2 F2 AB Ab aB ab

AB AABB AABb AaBB AaBb

MAS
Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

Select F2 plants that ab AaBb Aabb aaBb aabb
have Gene A and Gene B
 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 P2
Susceptible 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
PEDIGREE METHOD SINGLE-LARGE SCALE MARKER-
ASSISTED SELECTION (SLS-MAS)
P1 x P2
P1 x P2
F1
F1
Phenotypic
F2 screening F2 MAS
Plants space- Only desirable
planted in rows F3 lines planted
F3 for individual in field
plant selection F3
Families grown in
Families
progeny rows for
F4 grown in F4 selection.
progeny
rows for Pedigree selection
F5 selection. F5 based on local
Preliminary needs
yield trials.
F6 F6
Select single
plants.
Further
F7 F7
yield
trials
Multi-location testing, licensing, seed
Multi-location testing, F8 – F12
F8 – F12 increase and cultivar release
licensing, seed 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’)

Recurrent P1 (S) x P2 (R) Donor

Use when markers
Parent Parent
are not 100%
F1 (R) x P1 (S) accurate or when
phenotypic
BC1F1 phenotypes: R and S screening is more
expensive compared
MARKER-ASSISTED SELECTION (MAS)
to marker
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 …
genotyping
SAVE TIME & REDUCE
COSTS
PHENOTYPIC SELECTION
*Especially for quality traits*
Current status of molecular breeding

• A literature review
indicates thousands of
QTL mapping studies
but not many actual
reports of the
application of MAS in
breeding

• Why is this the case?

Some possible reasons to explain the low
impact of MAS in crop improvement
• Resources (equipment) not available
• Markers may not be cost-effective
• Accuracy of QTL mapping studies
• QTL effects may depend on genetic background
or be influenced by environmental conditions
• Lack of marker polymorphism in breeding material
• Poor integration of molecular genetics and
conventional breeding
A closer look at the examples of MAS indicates one
common factor:

• Most DNA markers have been developed for….

• In other words, not QTLs!! QTLs are

much harder to characterize!