1. THE USE OF
GENETIC MARKERS
IN PLANT BREEDING

2. Use of Molecular Markers
Clonal identity,
Family structure,
Population structure,
Phylogeny (Genetic Diversity)
Mapping
Parental analysis,
Gene flow,
Hybridisation

3. 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

4. Genetic Diversity

5. Clonal Identity early selection of the good allele fingerprints seeds,
plantlets

6. A linear order of genes or DNA fragments
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

7. It contains ordered overlapping cloned DNA fragment
Physical Mapping
It contains ordered overlapping cloned DNA fragment
The cloned DNA fragments are usually obtained using restriction enzyme digestion

8. Chromosomes with morphological Chromosomes with molecular
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. R r T t or
Chromosomes with morphological
marker alleles
RFLP1a
RFLP2a
RFLP4a
RFLP3a
SSR1a
SSR2a
RFLP1b
RFLP2b
RFLP4b
RFLP3b
SSR1b
SSR2b
Chromosomes with molecular
marker alleles

9. 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

10. Multigenic trait; ex: plant growth =Quantitative Trait Loci
Types of traits
Single gene trait: seed shape
Multigenic trait; ex: plant growth =Quantitative Trait Loci

11. Linkage groups

12. 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

13. 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

14. 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 individuals in the F2 showing each trait

15. → Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent
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

16. DNA markers can reliably predict phenotype
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

17. 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.

18. CONVENTIONAL PLANT BREEDINGx
Donor
Recipient F1
large populations consisting of thousands of plants F2
PHENOTYPIC SELECTION
Salinity screening in phytotron
Bacterial blight screening
Phosphorus deficiency plot
Field trials
Glasshouse trials

19. MARKER-ASSISTED BREEDINGP1 x P2
Susceptible
Resistant F1
large populations consisting of thousands of plants F2
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers

20. 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

21. Potential benefits from MAS
more accurate and efficient selection of specific genotypes
May lead to accelerated variety development
more efficient use of resources
Especially field trials
Crossing house
Backcross nursery

22. Overview of ‘marker genotyping’
(1) LEAF TISSUE SAMPLING
(2) DNA EXTRACTION
(3) PCR
(4) GEL ELECTROPHORESIS
(5) MARKER ANALYSIS

23. 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

24. 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

25. 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 individuals in the F2 showing each trait

26. → Near Isogenic Lines, Recombinant Inbreeds, Single Seed Decent
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

27. 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

28. Markers must be tightly-linked to target loci!
Ideally markers should be <5 cM from a gene or QTL
Marker A
QTL
5 cM
RELIABILITY FOR SELECTION
Using marker A only:
1 – rA = ~95%
Marker A
QTL
Marker B
5 cM
Using markers A and B:
1 - 2 rArB = ~99.5%
Using a pair of flanking markers can greatly improve reliability but increases time and cost

29. Markers must be polymorphic
RM84
RM296
P1 P2
P1 P2
Not polymorphic
Polymorphic!

30. DNA extractions LEAF SAMPLING Mortar and pestles
Porcelain grinding plates
LEAF SAMPLING
Wheat seedling tissue sampling in Southern Queensland, Australia.
High throughput DNA extractions “Geno-Grinder”

31. Agarose or Acrylamide gels
PCR-based DNA markers
Generated by using Polymerase Chain Reaction
Preferred markers due to technical simplicity and cost
PCR Buffer +
MgCl2 +
dNTPS +
Taq +
Primers +
DNA template
PCR
THERMAL CYCLING
GEL ELECTROPHORESIS
Agarose or Acrylamide gels

32. 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

33. MARKER ASSISTED BREEDING SCHEMES
Marker-assisted backcrossing
Pyramiding
Early generation selection
‘Combined’ approaches

34. 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
Target locus
RECOMBINANT SELECTION
BACKGROUND SELECTION
TARGET LOCUS SELECTION
FOREGROUND SELECTION
BACKGROUND SELECTION

35. 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

36. Select F2 plants that have Gene A and Gene B
Process of combining several genes, usually from 2 different parents, together into a single genotype
Breeding plan
Genotypes P1
Gene A x P1
Gene B
P1: AAbb x
P2: aaBB F1
Gene A + B
F1: AaBb F2 F2 AB Ab aB ab
AABB
AABb
AaBB
AaBb
AAbb
Aabb
aaBB
aaBb
aabb
MAS
Select F2 plants that have Gene A and Gene B

37. 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

38. large populations (e.g. 2000 plants)x P2
Susceptible
Resistant F1 F2
large populations (e.g plants)
MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes
MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes

39. SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS)
PEDIGREE METHOD
P1 x P2 F1 F2 F3
MAS
SINGLE-LARGE SCALE MARKER-ASSISTED SELECTION (SLS-MAS) F4
Families grown in progeny rows for selection.
Pedigree selection based on local needs F6 F7 F5
F8 – F12
Multi-location testing, licensing, seed increase and cultivar release
Only desirable F3 lines planted in field
P1 x P2 F1
Phenotypic screening F2
Plants space-planted in rows for individual plant selection F3
Families grown in progeny rows for selection. F4 F5
Preliminary yield trials. Select single plants. F6
Further yield trials F7
Multi-location testing, licensing, seed increase and cultivar release
F8 – F12
Benefits: breeding program can be efficiently scaled down to focus on fewer lines

40. Combined approaches
In some cases, a combination of phenotypic screening and MAS approach may be useful
To maximize genetic gain (when some QTLs have been unidentified from QTL mapping)
Level of recombination between marker and QTL (in other words marker is not 100% accurate)
To reduce population sizes for traits where marker genotyping is cheaper or easier than phenotypic screening

41. ‘Marker-directed’ phenotyping
(Also called ‘tandem selection’)
Recurrent
Parent
P1 (S) x P2 (R)
Donor
Parent
Use when markers are not 100% accurate or when phenotypic screening is more expensive compared to marker genotyping
F1 (R) x P1 (S)
BC1F1 phenotypes: R and S
MARKER-ASSISTED SELECTION (MAS) …
SAVE TIME & REDUCE COSTS
PHENOTYPIC SELECTION
*Especially for quality traits*