the use of genetic markers in plant breeding
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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