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 DiversityGenetic 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 DiversityGenetic Diversity

early selectionof the good allele

seeds,plantlets

fingerprints

Clonal Identity

MappingThe determination of the position and

relative distances of gene on chromosome by means of their linkage

Genetic mapA linear arrangement of genes or genetic markers

obtained based on recombination

Physical mapA linear order of genes or DNA fragments

Physical MappingPhysical Mapping

It contains ordered overlapping cloned DNA fragment

The cloned DNA fragments are usually obtained using restriction

enzyme digestion

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.

R r

T t

or

Chromosomes with morphological marker alleles

RFLP1aRFLP2a

RFLP4a

RFLP3a

SSR1a

SSR2a

RFLP1b

RFLP2b

RFLP4b

RFLP3b

SSR1b

SSR2b

Chromosomes with molecular marker alleles

Genetic Maps

QTL MappingQTL Mapping

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 (Quantitative Trait Loci)QTL (Quantitative Trait Loci)

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

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

Trait Loci

Types of traits

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 MarkersDeveloping 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 MarkerConduct 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

MASMAS Marker assisted selection

The use of DNA markers that are tightly-linked to target loci as a

substitute for or to assist phenotypic screening

DNA markers can reliably predict phenotype

Assumption

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.

F2

P2

F1

P1 x

large populations consisting of thousands of plants

PHENOTYPIC SELECTION

Field trialsGlasshouse trials

DonorRecipient

CONVENTIONAL PLANT BREEDING

Salinity screening in phytotron

Bacterial blight screening Phosphorus deficiency plot

F2

P2

F1

P1 x

large populations consisting of thousands of plants

Resistant

Susceptible

MARKER-ASSISTED SELECTION (MAS)

MARKER-ASSISTED BREEDING

Method whereby phenotypic selection is based on DNA markers

Advantages of MASAdvantages of MAS Simpler method compared to Simpler method compared to

phenotypic screeningphenotypic screening• Especially for traits with laborious Especially for traits with laborious

screeningscreening• May save time and resourcesMay save time and resources

Selection at seedling stageSelection at seedling stage• Important for traits such as grain qualityImportant for traits such as grain quality• Can select before transplanting in rice Can select before transplanting in rice

Increased reliabilityIncreased reliability• No environmental effectsNo environmental effects• Can discriminate between homozygotes Can discriminate between homozygotes

and heterozygotes and select single and heterozygotes and select single plantsplants

Potential benefits from MASPotential benefits from MAS more accurate and more accurate and

efficient selection efficient selection of specific of specific genotypesgenotypes• May lead to May lead to

accelerated variety accelerated variety development development

more efficient use more efficient use of resourcesof resources• Especially field Especially field

trialstrials

Crossing house

Backcross nursery

(1) LEAF TISSUE SAMPLING

(2) DNA EXTRACTION

(3) PCR

(4) GEL ELECTROPHORESIS

(5) MARKER ANALYSIS

Overview of ‘marker genotyping

’

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 MarkersDeveloping 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 MarkerConduct 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 Considerations for using DNA markers in plant breedingmarkers in plant breeding

Technical methodologyTechnical methodology• simple or complicated?simple or complicated?

ReliabilityReliability Degree of polymorphismDegree of polymorphism DNA quality and quantity DNA quality and quantity

requiredrequired Cost**Cost** Available resourcesAvailable resources

• Equipment, technical expertiseEquipment, technical expertise

Markers must be Markers must be tightly-linked to target loci!tightly-linked to target loci!

Ideally markers should be <5 cM from a gene or Ideally markers should be <5 cM from a gene or QTLQTL

• Using a pair of flanking markers can greatly improve reliability but increases time and cost

Marker A

QTL5 cM

RELIABILITY FOR SELECTION

Using marker A only:

1 – rA = ~95%Marker A

QTL

Marker B

5 cM 5 cM

Using markers A and B:

1 - 2 rArB = ~99.5%

Markers Markers mustmust be polymorphic be polymorphic

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

RM84 RM296

P1 P2

P1 P2

Not polymorphic Polymorphic!

DNA extractionsDNA extractions

LEAF SAMPLING

Porcelain grinding plates

High throughput DNA extractions “Geno-Grinder”

Mortar and pestles

Wheat seedling tissue sampling in Southern Queensland,

Australia.

PCR-based DNA markersPCR-based DNA markers Generated by using Polymerase Chain Reaction Preferred markers due to technical simplicity and cost

GEL ELECTROPHORESIS

Agarose or Acrylamide gels

PCR

PCR Buffer +

MgCl2 +

dNTPS +

Taq +

Primers +

DNA template

THERMAL CYCLING

Useful when the gene(s) of interest is difficult to select:

1. Recessive Genes2. Multiple Genes for Disease Resistance3. Quantitative traits4. Large genotype x environment interaction

Marker Assisted Selection

MARKER ASSISTED MARKER ASSISTED BREEDING SCHEMESBREEDING SCHEMES

1.1. Marker-assisted Marker-assisted backcrossingbackcrossing

2.2. PyramidingPyramiding

3.3. Early generation selectionEarly generation selection

4.4. ‘‘Combined’ approachesCombined’ approaches

Marker-assisted backcrossing Marker-assisted backcrossing (MAB)(MAB)

MAB has several advantages over MAB has several advantages over conventional backcrossing:conventional backcrossing:• Effective selection of target lociEffective selection of target loci• Minimize linkage dragMinimize linkage drag• Accelerated recovery of recurrent parentAccelerated recovery of recurrent parent

1

2 3 4

Target locus

1

2 3 4

RECOMBINANT SELECTION

1

2 3 4

BACKGROUND SELECTION

TARGET LOCUS SELECTION

FOREGROUND SELECTION BACKGROUND SELECTION

Gene PyramidingGene Pyramiding Widely used for combining multiple

disease resistance genes for specific races of a pathogen

Pyramiding is extremely difficult to achieve using conventional methodsConsider: phenotyping a single plant for multiple forms of seedling resistance – almost impossible

Important to develop ‘durable’ disease resistance against different races

F2

F1

Gene A + B

P1

Gene Ax P1

Gene B

MAS

Select F2 plants that have Gene A and

Gene B

Genotypes

P1: AAbb P2: aaBB

F1: AaBb

F2AB Ab aB ab

AB AABB AABb AaBB AaBb

Ab AABb AAbb AaBb Aabb

aB AaBB AaBb aaBB aaBb

ab AaBb Aabb aaBb aabb

Process of combining several genes, usually from 2 different parents, together into a single genotype

x

Breeding plan

Early generation MASEarly generation MAS MAS conducted at F2 or F3 stageMAS conducted at F2 or F3 stage Plants with desirable genes/QTLs are Plants with desirable genes/QTLs are

selected and alleles can be ‘fixed’ in the selected and alleles can be ‘fixed’ in the homozygous statehomozygous state• plants with undesirable gene combinations plants with undesirable gene combinations

can be discardedcan be discarded Advantage for later stages of breeding Advantage for later stages of breeding

program because resources can be used program because resources can be used to focus on fewer linesto focus on fewer lines

F2

P2

F1

P1 x

large populations (e.g. 2000 plants)

Resistant

Susceptible

MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes

MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes

P1 x P2

F1

PEDIGREE METHOD

F2

F3

F4

F5

F6

F7

F8 – F12

Phenotypic screening

Plants space-planted in rows for individual plant selection

Families grown in progeny rows for selection.

Preliminary yield trials. Select single plants.

Further yield trials

Multi-location testing, licensing, seed increase and cultivar release

P1 x P2

F1

F2

F3

MAS

SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)

F4Families grown in progeny rows for selection.

Pedigree selection based on local needs

F6

F7

F5

F8 – F12Multi-location testing, licensing, seed increase and cultivar release

Only desirable F3 lines planted in field

Benefits: breeding program can be efficiently scaled down to focus on

fewer lines

Combined approachesCombined approaches In some cases, a combination of In some cases, a combination of

phenotypic screening phenotypic screening andand MAS MAS approach may be usefulapproach may be useful

1.1. To maximize genetic gain (when some To maximize genetic gain (when some QTLs have been unidentified from QTL QTLs have been unidentified from QTL mapping)mapping)

2.2. Level of recombination between marker Level of recombination between marker and QTL (in other words marker is not and QTL (in other words marker is not 100% accurate)100% accurate)

3.3. To reduce population sizes for traits To reduce population sizes for traits where marker genotyping is cheaper or where marker genotyping is cheaper or easier than phenotypic screeningeasier than phenotypic screening

‘‘Marker-directed’ phenotypingMarker-directed’ phenotyping

BC1F1 phenotypes: R and S

P1 (S) x P2 (R)

F1 (R) x P1 (S)

RecurrentParent

DonorParent

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 …

SAVE TIME & REDUCE COSTS

*Especially for quality traits*

MARKER-ASSISTED SELECTION (MAS)

PHENOTYPIC SELECTION

(Also called ‘tandem selection’)

Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping