Marker-assisted selection (MAS) uses DNA markers linked to traits of interest to assist plant breeders in selecting desirable plants. MAS has advantages over phenotypic selection like enabling selection at early stages. MAS breeding schemes include marker-assisted backcrossing to introgress traits while minimizing linkage drag, and pyramiding to combine multiple genes/QTLs. Case studies demonstrate using MAS to develop rice varieties with submergence tolerance and improve yield traits. However, limitations include inconsistent QTL-marker associations across environments and difficulties evaluating complex trait genetics like epistasis. Future work aims to optimize MAS efficiency and integration with plant breeding.

2. MAS refers to the use of DNA markers that are
tightly linked to target loci as a substitute for or to
assist phenotypic screening
Marker assisted selection (MAS)
Assumption: DNA markers can reliably predict phenotype

3. Multifactorial traits are determined by multiple genetic and
environmental factors acting together
Genetic architecture of a complex trait = specific effects and
combined interactions of all genetic and environmental factors
Quantitative traits = phenotypes differ in quantity rather
than type (such as height)
Variation in genotype can be eliminated by studying inbred
lines = homozygous for most genes, or F1 progeny of inbred
lines = uniformly heterozygous
Complete elimination of environmental variation is
impossible
Multifactorial = complex traits = quantitative traits

4. Considerations for using DNA
markers in plant breeding
1. Technical methodology
simple or complicated?
1. Reliability
2. Degree of polymorphism
3. DNA quality and quantity required
4. Cost
5. Available resources
Equipment, technical expertise
Collard et al., 2008

5. F2
P2
F1
P1
x
Large populations consisting of
thousands of plants
PHENOTYPIC SELECTION
Field trialsGlasshouse trials
DonorRecipient
Salinity screening in phytotron Bacterial blight screening Phosphorus deficiency plot

6. F2
P2
F1
P1 x
large populations consisting of
thousands of plants
ResistantSusceptible
MARKER-ASSISTED SELECTION (MAS)
Method whereby phenotypic selection is based on DNA markers

7. Advantages of MAS
Simpler method compared to phenotypic screening
Selection at seedling stage
Increased reliability
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
Collard et al., 2008

8. MAS BREEDING SCHEMES
1. Marker-assisted backcrossing
2. Pyramiding
3. Early generation selection
4. ‘Combined’ approaches

9. 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
1 2 3 4
RECOMBINANT SELECTION
1 2 3 4
BACKGROUND SELECTIONTARGET LOCUS SELECTION
FOREGROUND SELECTION BACKGROUND SELECTION

10. P1 x F1
P1 x P2
CONVENTIONAL BACKCROSSING
BC1
VISUAL SELECTION OF BC1 PLANTS THAT MOST CLOSELY
RESEMBLE RECURRENT PARENT
BC2
MARKER-ASSISTED BACKCROSSING
P1 x F1
P1 x P2
BC1
USE ‘BACKGROUND’ MARKERS TO SELECT PLANTS THAT HAVE
MOST RP MARKERS AND SMALLEST % OF DONOR GENOME
BC2

11. 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

12. F2
F1
Gene A + B
P1
Gene A
x P1
Gene B
MAS
Select F2 plants that have
Gene A and Gene B
Genotypes
P1: AAbb P2: aaBB
F1: AaBb
Process of combining several genes, usually from two different
parents, together into a single genotype
x
Breeding plan
Hittalmani et al. (2000) and Liu et al. (2000)

13. 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
Ribaut & Betran. (1999)

14. 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)
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
Benefits: breeding program can be efficiently
scaled down to focus on fewer lines

15. 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

16. Local cultivars from Vietnam - OM 1490/ OMCS 2000 recurrent parents
IR -64 sub-1- donor parent for submergence tolerance
CASE STUDY- 1

17. OM 1490/
OMCS 2000
X IR-64 sub-1
X OM 1490/
OMCS 2000
BC1F1 X OM 1490/
OMCS 2000
BC2F1Selfing
BC2F2BC2F3
Selfing
Yield and yield
components tested
Lang et al, 2011
Screening for
submergence resistance
F1
Development of submergence tolerance rice varieties

18. PCR products of BC2F2 population from OM 1490 / IR 64 sub-1 at locus RM
23804 on chromosome number 9 (1 IR64-Sub1; 2 OM1490; 1 and 125 BC2F2 progenies )
Lang et al, 2011

19. Yield and yield components of rice genotypes tested in BC2F3 at CLRRI
Lang et al, 2011

20. Preparation of CSSL by using to contrast parents for yield trait, genotyping
and phenotyping
Generation of NIL lines for the specific QTLs, Pyramiding of NIL QTL lines to
see the combined effect of the both the QTLs identified
Analysis for additive , dominant and epistatic interactions
CASE STUDY- 2

21. Strategies for development of CSSL
(31)BIL
Genotyping with 236 RFLP markers
Genotyping with 236 RFLP markers
30 lines
Genotyped with 116 SSR
markers distributed uniformly
at an interval of 2.6 Mb
Ando et al., 2008

22. Graphical representation of genotype of whole chromosomes in
the
CSSLs developed
Ando et al., 2008

23. Ando et al., 2008
Chromosomal locations of QTLs for panicle architecture
(QTLs detected in 2004 and 2005)

24. Development of QTL -NILs
Ando et al., 2008
Sasanishiki X BIL-8
F1 X sasanishiki
BC1F3
NIL for SBN1
Sasanishiki X BIL-21
F1 X sasanishiki
BC1F1 X sasanishiki
BC1F1
BC2F1
BC2F3
NIL for PBN 6X
NIL (SBN-1+ PBN-6)

25. Ando et al., 2008
Phenotypic performance
of QTL -NILs

26. Wild species used- Oryza rufipogon
V20A- recurrent female parent
V20B- maintainer line
Ce 64- restorer
V20A X Ce 64 – F1 hybrid shows strong
vigour, used for comparing the BC2 test cross
progeny
PCA of 34 wild accessions and15 accessions from cultivated species
CASE STUDY- 3

27. V20A X O. rufipogon
(IRGC-105491)
V 20 BXF1
BC1F1 V 20 BX
BC2F1 X Ce 64
BC2 test cross progeny
300 test cross families
52plants 10 plants
3000plants 300plants
QTL analysis
Field trail and trait evaluation
Xiao et al. (1998)
Identification of trait improving Quantitative trait alleles from a wild
rice relative Oryza rufipogon (AB-QTL )

28. Xiao et al. (1998)
Frequency distribution of phenotypic for different traits in BC2 test cross progeny

29. Xiao et al. (1998)
Frequency distribution of phenotypic for different traits in BC2 test cross progeny

30. Azhul X Spontaneum.
I
F1Azhul X
BC1F1 X Azhul
BC2F1 X Azhul
BC3F1
BC3F3
Genotyping and QTL analysis
Eshghi et al., 2013
Hull less variety Hulled wild relative
CASE STUDY- 4

31. Correlation matrix of the traits analyzed in the Azhul X Spontanum.I
population
Eshghi et al., 2013

32. QTLs for yield and yield components detected in the BC3 population from Azhul x
Spontaneum

33. Identification of limited number of major ‘players’ (QTLs)
controlling specific traits
Inadequacies / experimental deficiencies in QTL analysis
leading to either over estimation or under estimation of the
number of effects of QTLs
Lack of universally valid QTL/ marker associations
applicable over different sets of breeding material
Strong QTL X environmental interaction
Difficulty in precisely evaluating epistatic effects
Limitations on efficient utilization of QTL mapping
information in plant breeding through MAS

34. Future challenges
Improved cost-efficiency
Optimization, simplification of
methods and future innovation
Design of efficient and effective
MAS strategies
Greater integration between
molecular genetics and plant
breeding
Data management