MARKER ASSISTED SELECTION IN CROP IMPROVEMENT

2. Doctoral Seminar Presentation on
“MARKER ASSISTED SELECTION IN CROP IMPROVEMENT”
Presented by
Mr. PAWAR VINOD SHRIPATI
Reg.No.2017/07

3. INTRODUCTION
MARKER ASSISTED SELECTION THEORY AND
PRACTICE
MAS BREEDING SCHEMES
CASE STUDIES
CURRENT STATUS OF MAS

4. • Conventional breeding has got tremendous success
towards feeding world
• But future demands continuous development of
new crop varieties to suitable to fight diverse
problems in less time frame
• To address this problem MAS can be
supplementary to conventional breeding
programme

6. Marker assisted selection (MAS) refers to 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

7. F2
P2
F1
P1 x
large populations consisting of
thousands of plants
DonorRecipient
Salinity screening in phytotron Bacterial blight screening
Phosphorus deficiency plot

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

9. 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

10. • 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

11. 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

12. • Technical methodology
– simple or complicated?
• Reliability
• Degree of polymorphism
• DNA quality and quantity required
• Cost**
• Available resources
– Equipment, technical expertise

13. • Ideally markers should be <5 cM from a gene or QTL
• Using a pair of flanking markers can greatly improve reliability but increases time
and cost
Marker A
QTL
5 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%

14. 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!

16. • 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
SELECTION
TARGET LOCUS
SELECTION
FOREGROUND SELECTION

17. • Selection for target gene or
QTL
• Useful for traits that are difficult
to evaluate
• Also useful for recessive genes
1 2 3 4
Target locus
TARGET LOCUS
SELECTION
FOREGROUND SELECTION

18. Donor/F1 BC1
c
BC3 BC10
TARGET
LOCUS
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME
TARGET
LOCUS
LINKEDDONOR
GENES
• Large amounts of donor chromosome remain even after many backcrosses
• Undesirable due to other donor genes that negatively affect agronomic
performance

19. Conventional backcrossing
Marker-assisted backcrossing
F1 BC1
c
BC2
c
BC3 BC10 BC20
F1
c
BC1 BC2
• Markers can be used to greatly minimize the amount
of donor chromosome….but how?
TARGET
GENE
TARGET
GENE
Ribaut, J.-M. & Hoisington, D. 1998 Marker-assisted selection:
new tools and strategies. Trends Plant Sci. 3, 236-239.

20. 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

21. • Use flanking markers to select
recombinants between the
target locus and flanking
marker
• Linkage drag is minimized
• Require large population sizes
– depends on distance of flanking
markers from target locus)
• Important when donor is a
traditional variety
RECOMBINANT
SELECTION
1 2 3 4

22. • Use unlinked markers to
select against donor
• Accelerates the recovery of
the recurrent parent genome
• Savings of 2, 3 or even 4
backcross generations may
be possible
1 2 3 4
BACKGROUND
SELECTION

24. Cont…

25. Conclusion of the Study...
Selection criteria I : (High oil content with high oleic acid and low
linoleic and palmitic acid.) 17 ILs MABC and 10 MAS were selected. ILs
includes, 3 lines from MABC Cross-I, 4 lines from MABC Cross-II and
10 lines from MABC Cross-III.
The oil content in these lines was high and varied from 53.0 to 57.9%.
Based on Selection Cri-teria II: 18 ILs from MABC crosses, which
included one line from MABC Cross-I, 8 lines from MABC Cross-II and
9 lines from MABC Cross-III, and 10 ILs from MAS Cross were
selected.
The oil content in these lines varied from 42.4 to 49.9%.
Low oil content peanuts are needed for table purposes, confections and
several other food uses.
The oleic acid content in donor parent, Sunoleic 95R, was 78.0% and
among the selected ILs with high/low oil content, it varied from 62 to
83%.
Besides, all the ILs, selected under both the selection criteria, recorded a
decrease in linoleic acid by 0.4–1.0 folds, and palmitic acid by 0.1–0.6 as
compared to recurrent parents.
Reduced linoleic and palmitic acid contents have additional health
benefits to consumers.

26. • 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

27. 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
F2
AB 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
Liu et al. (2000). Molecular marker-facilitated pyramiding of different genes for powdery mildew resistance in wheat. Plant
Breeding 119: 21-24.

29. Samba Mahsuri (BPT 5204) x SS 1113
F1 x Samba Mahsuri (used as female)
BC1F1
11/145 plants were found to be triple heterozygotes for R gene linked markers. Three
STS markers, pTA248 is 0.2 cM from Xa21, (Ronald et al. 1992), the RG136 marker
is 1.5 cM from Xa13 (Zhang et al. 1996) and the RG556 marker is 0.1 cM from Xa5
(Yoshimura et al. 1995).) 139 rice microsatellite markers that were screened, 50 were
found to be polymorphic between parents.
BC4F1
BC4F2
(25 lines have 3 gene.)
Glass house evaluation for their resistance to BB
BC4F6
(four of the three gene pyramid lines along with donor and recipient parents were
evaluated under AICRIP in year 2005 at 10 selected locations across India.)
Recipient Donor

30. Table 1 Effect of bacterial blight (BB) on yield of Samba Mahsuri and
three-gene pyramid lines
Line
Line Yield (kg/ha)
under BB free
condition
Yield (kg/ha)
under BB
infection
% of yield
reduction due to
BB
Samba Mahsuri 6850 ± 403 5250 ± 296 23.36
B170 6613 ± 451 6432 ± 498 2.72
B189 7205 ± 623 6913 ± 447 4.05
B197 6932 ± 455 6731 ± 405 2.89
B226 6695 ± 432 6525 ± 393 2.54
Conclusion : lines retain the excellent grain and cooking qualities of
Samba Mahsuri without compromising the yield under BB infection
conditions.
Raman M. Sundaram et al. 2008

31. • 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
References:
Ribaut & Betran (1999). Single large-scale marker assisted selection (SLS-MAS). Mol Breeding 5: 21-24.

32. F2
P2
F1
P1 x
large populations (e.g. 2000 plants)
ResistantSusceptible
MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes

33. 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

35. Sariaso 09 x BTx642 (B35)
Recipient parent Donor parent
(Stg1, Stg2, Stg3, Stg4 and StgB)
Hybridity checked F1 x Sariaso09
BCF1
Foreground selection with 17 SSR Markers and Background selection with 18
SSR markers. 83 % recovery of recipient parent.
Subsequent selection in generation by phenotyping.
Conclusions and applications of findings:
Among BC1F1 generation, 30 progenies had
incorporated at least one stay-green QTL.
18 progenies had 5 stay green QTL. (Stg1, Stg2, Stg3, Stg4 and StgB)
5 progenies had incorporated 3 stay green QTL.
4 plants incorporated double QTL (Stg3 and StgB)
Two of the introgression lines had high levels of the recurrent parents’
genomes and constitute some promising lines to develop drought tolerant
varieties.

36. • 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

37. BC1F1 phenotypes: R and S
P1 (S) x P2 (R)
F1 (R) x P1 (S)
Recurrent
Parent
Donor
Parent
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
References:
Han et al (1997). Molecular marker-assisted selection for malting quality traits in barley. Mol Breeding 6: 427-437.

38. TDK1 is a popular rice variety from the Lao PDR. Originally developed
for irrigated conditions, this variety suffers a high decline in yield under
drought conditions.
Studies have identified three quantitative trait loci (QTLs) for grain yield
under drought conditions, qDTY3.1, qDTY6.1, and qDTY6.2, that show
a high effect in the background of this variety.
We report here the pyramiding of these three QTLs with SUB1 that
provides 2–3 weeks of tolerance to complete submergence, with the aim to
develop drought- and submergence-tolerant near-isogenic lines (NILs) of
TDK1.

39. Mapping population TDK 1
TDK 1 x IR55419-04 (qDTY 3.1, qDTY 6.1 and qDTY 6.2)
Introgression of Sub 1
F1 x TDK 1
BC1F3:5 (qDTY 3.1, qDTY 6.1 and qDTY 6.2) x TDK 1 – Sub 1
17 BC1F5 lines with 3 QTL identified. BC2F1 (658 Plants)
BC2F2 (10,000 Plants)
This generation onward a tandem phenotypic selection for TDK 1 plant type.
5700 selected plant then genotyped with the foreground selection. Plant with different
combinations of QTL and Sub 1 were identified.
BC2F3 (834 lines developed from selected plants for further purification and testing.)
Population screened under drought stress and non stress condition.
(228 single plant selected for BC2F4)
Screened under drought stress, submergence condition. (44 lines advanced to the BC2F5)
BC2F6 (105 Panicles similar to TDK1 or TDK 1 Sub 1selected and screened under drought and
submergence condition)
BC2F7 (42 Lines selected and screened)
BC2F8 (29 NILs Testing for RYT. )
Conclusion : NIL TDK1-Sub1 Ready for release as a drought and submergence
tolerance variety.

41. • 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

42. • Cost-efficiency has rarely been
calculated but MAS is more
expensive for most traits
– Exceptions include quality traits
• Determined by:
– Trait and method for phenotypic
screening
– Cost of glasshouse/field trials
– Labour costs
– Type of markers used

43. • Reliable phenotypic data critical!
– Multiple replications and environments
• Confirmation of QTL results in independent
populations
• “Marker validation” must be performed
– Testing reliability for markers to predict phenotype
– Testing level of polymorphism of markers
• Effects of genetic background need to be
determined

44. • Large ‘gaps’ remain between marker
development and plant breeding
– QTL mapping/marker development have been
separated from breeding
– Effective transfer of data or information
between research institute and breeding station
may not occur
• Essential concepts in may not be understood
by molecular biologists and breeders (and
other disciplines)

45. 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

46. Achievements of MAS in India
1. Three BB resistance genes (Xa5, Xa13, Xa21) introgress
into the rice variety Sambha Mahsuri (BPT-5204)
released in India as “Improved Sambha Mahsuri”
2. BB resistance genes (Xa13, Xa21) introgress into the rice
variety Pusa Basmati 1 (PB 1) and released as
“Improved Pusa Basmati (IPB 1)”
3. Submergence tolerance variety: Swarna- Sub 1 and
Sambha Mahsuri- Sub1 Show increased submergence
tolerance.
4. Improved Pusa RH10. resistant to BB and blast.
5. Maize : extra early QPM hybrid “Vivek QPM 9”
6. Pearl millet a downey mildew (Sclerospora graminicola)
resistant hybrid, “HHB 67-2”, has been developed by
MAS.

47. Referances…
• Collard, Bertrand CY, and David J. Mackill. "Marker-assisted selection: an approach for
precision plant breeding in the twenty-first century." Philosophical Transactions of the
Royal Society B: Biological Sciences 363.1491 (2008): 557-572.
• Dixit, Shalabh, et al. "Combining drought and submergence tolerance in rice: marker-
assisted breeding and QTL combination effects." Molecular Breeding 37.12 (2017): 143.
• Janila, Pasupuleti, et al. "Molecular breeding for introgression of fatty acid desaturase
mutant alleles (ahFAD2A and ahFAD2B) enhances oil quality in high and low oil
containing peanut genotypes." Plant Science 242 (2016): 203-213.
• Liu, J., et al. "Molecular marker‐facilitated pyramiding of different genes for powdery
mildew resistance in wheat." Plant Breeding 119.1 (2000): 21-24.
• Ouedraogo, Nofou, et al. "Incorporation of stay-green Quantitative Trait Loci (QTL) in
elite sorghum (Sorghum bicolor L. Moench) variety through marker-assisted selection at
early generation." Journal of Applied Biosciences 111.1 (2017): 10867-10876.
• Singh, B. D., and Singh A.K., Marker-assisted plant breeding: principles and practices.
New Delhi, India: Springer, 2015.
• Tanksley & Nelson (1996). Advanced backcross QTL analysis: a method for the
simultaneous discovery and transfer of valuable QTLs from unadapted germplasm into
elite breeding lines. Theor. Appl. Genet. 92: 191-203