This document discusses the use of marker-assisted selection (MAS) in plant breeding. It begins by outlining some key challenges in plant breeding, then describes how MAS can accelerate the breeding cycle by allowing selection at early generations. It provides details on different types of MAS, including marker-assisted backcrossing, pyramiding of multiple genes, and early generation selection. Examples are given of MAS being used to introgress submergence tolerance and salinity tolerance genes into rice varieties. The document also discusses some reasons for the low impact of MAS to date, such as insufficient linkage between markers and traits.

1. “SMART breeding: A non-invasive
biotechnology alternative to
genetic engineering of plant
varieties”
1
Pavan. R
Ph. D Scholar
Department of Genetics and Plant Breeding
University of Agricultural Sciences
Bengaluru-65

2. 2

3. 3

4. Key challenges
Feeding the world within the carrying capacity of
planet earth (2x more with 2x less)
Improve food security, safety and quality
Increase the production per ha
Reduce input at the same time
Use biomass for bio-fuels and green chemistry while
securing food production
4

5. Accelerating The Breeding Cycle
5

6. Breeder’s equation
6

7. Selection Intensity
 Large F2 populations
 Big screening nurseries
 Many crosses / populations
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8. Selection Accuracy
Many replicated trials
National/International trials
8

9. Genetic Variance
 Bring in new genes not present in current program
Nachum Kedar, Israeli Scientist
9

10. Years per Cycle
10

11. Running short of time……….!!
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12. Use of molecular markers in selection
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13. How molecular markers server our purpose?
Simpler than phenotypic screening, which can save
time, resources and effort
Selection can be carried out at the seedling stage
Single plants can be selected
Shortens the breeding cycle
Selection of drought tolerant plant without drought
No question of biosafety and bioethics
13

14. Defined as DNA sequence with a known location on
a chromosome.
Molecular marker
Qualities of a Suitable molecular marker are:
1) Must be polymorphic
2) Co-dominant inheritance
3) Randomly and frequently distributed throughout the
genome
4) Easy and cheap to detect
5) Easily reproducible
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15. FIRST GENERATION DNA MARKERS
Year Acronym Nomenclature Reference
1974 RFLP Restrition Fragment Length Polymorphism Grodzicker et al. (1974)
1985 VNTR Variable Number Tandem Repeats Jeffreys et al. (1985)
1989 SSCP Single Stranded Conformational Polymorphism Orita et al. (1989)
1989 STS Sequence Tagged Site Olsen et al. (1989)
SECOND GENERATION DNA MARKERS
1990 RAPD Randomly Amplified Polymorphic DNA Williams et al. (1990)
1992 CAPS Cleaved Amplified Polymorphic Sequence Akopyanz et al. (1992)
1992 SSR Simple Sequence Repeats Akkaya et al. (1992)
1993 SCAR Sequence Characterized Amplified Region Paran and Michelmore (1993)
NEW GENERATION DNA MARKERS
1994 ISSR Inter Simple Sequence Repeats Zietkiewicz et al (1994)
1994 SNP Single Nucleotide Polymorphisms Jordan and Humphries (1994)
1995 AFLP Amplified Fragment Length Polymorphism Vos et al. (1995)
1996 ISTR Inverse Sequence-Tagged Repeats Rhode (1996)
1997 S-SAP Sequence-Specific Amplified Polymorphism Waugh et al. (1997)
1998 RBIP Retrotransposon Based Insertional Polymorphism Flavell et al. (1998)
1999 REMAP Retrotransposon-Microsatellite Amplified Polymorphism Kalendar et al. (1999).
2001 SRAP Sequence-related amplified polymorphism Li and Quiros (2001)
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16. Different types of molecular marker profiles
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17. Marker assisted selection (MAS):
Defined as Phenotype is selected based on the
genotype of the marker.
 Reliability: Markers should be tightly linked to target
loci, preferably less than 5 cM genetic distance. DNA
quantity and quality.
 DNA quality and quantity
 Level of polymorphism: it should discriminate
between different genotypes
 Cost
Assumption
17

18. F2
P2
F1
P1 x
large populations consisting of thousands
of plants
ResistantSusceptible
MARKER-ASSISTED SELECTION (MAS)
MARKER-ASSISTED BREEDING
Method whereby phenotypic selection is based on DNA markers
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19. Marker development
‘pipeline’
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20. APPLICATIONS OF MAS IN PLANT BREEDING
1. Marker assisted evaluation of breeding material
2. Marker-assisted backcrossing
3. Marker-assisted pyramiding
4. Early generation marker-assisted selection and
5. Combined marker-assisted selection
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21. 1. Marker-assisted evaluation of breeding material
 Cultivar identity/assessment of ‘purity
 Assessment of genetic diversity and parental selection
 Study of heterosis
 Identification of genomic regions under selection
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22. 2. Marker-assisted backcrossing (MAB)
Advantages over conventional backcrossing:
◦ Minimize linkage drag
◦ Accelerated recovery of recurrent parent- Young & Tanksley (1989)
◦ Effective selection of target loci- Tanksely (1983)
FOREGROUND
SELECTION
BACKGROUND SELECTION
22

23. Foreground selection
Proposed by Tanksely (1983)
 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
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24. Donor/F1 BC1
c
BC3 BC10
TARGET
LOCUS
RECURRENT PARENT
CHROMOSOME
DONOR
CHROMOSOME
TARGET
LOCUS
LINKEDDONOR
GENES
Concept of ‘linkage drag’
• Large amounts of donor chromosome remain even after
many backcrosses
•Undesirable due to other donor
genes that negatively affect
agronomic performance
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25. • Markers can be used to greatly minimize the
amount of donor chromosome….but how?
Marker-assisted backcrossing
F1
c
BC1 BC2
TARGET
GENE
Conventional backcrossing
F1 BC1
c
BC2
c
BC3 BC10 BC20
TARGET
GENE
Reduces the
number of
breeding
cycles
(Ribaut and Hoisington, 1998 )
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26. Recombinant selection
 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
RECOMBINANT
SELECTION
1 2 3 4
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27. Background selection
 Proposed by Young and Tanksley
(1989) and termed by Hospital and
Charcosset (1997)
 Accelerates the recovery of the
recurrent parent genome
 Savings of 3 to 4 backcross
generations
1 2 3 4
BACKGROUND
SELECTION
27

28. Ideal Markers must be
Ideally markers should be <5 cM from a gene or QTL
tightly-linked to target loci!
Marker A
QTL
5 cM
RELIABILITY FOR
SELECTION
Using marker A only:
~95%
rA
QTL
Marker B
4 cM
Using marker B only
~96%
rB
Marker A
QTL
Marker B
5 cM 4 cM
Using markers A and B:
~99.5%
rA rB
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29. Markers must be polymorphic
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
P1 P2
P1 P2
Not polymorphic Polymorphic!
29

30.  Regularly affects 15 million hectares or more of rainfed
lowland rice areas in South and Southeast Asia.
 An economic loss of up to one billion US dollars annually has
been estimated
 A major QTL, Sub1 was fine mapped on chromosome 9 in
FR13A cultivar, IR49830-7 (Donor parent)
 Swarna (Recipient parent), widely grown cultivar in India and
Bangladesh
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31. Foreground selection &
recombination selection
RM219 – 3.4 cM/Sub1/ RM464A -0.7 cM,
RM316- 1.5 cM
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32. (Neeraja et al., 2007)32

33. Screening for submergance tolerance
Fourteen- day-old seedlings were submerged for 14 days(BC1F2,
BC2F2 and BC3F2). The survival of plants was scored 14 days
after de-submergence (calculated as a percentage) for
confirmation of the presence of the Sub1 locus.
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34. Introgressed fragment containing Sub1 in a selected
BC3F2 plant
BC3F2 plant (No. 227-9-407) Selected BC2F2 plant No. 246-237
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35. 35

36. Submergence tolerance of Swarna-Sub1 (BC2 progeny)
36

37.  Salinity is the most common abiotic stresses affects rice
growth in all stages of crop, leads to the reduction in yield
 Major salinity tolerance QTL, named Saltol, which responsible
for seedling-stage salinity tolerance was identified on the
short arm of chromosome 1
 Used 368 SSR markers, 89 were polymorphic (8 in Saltol locus)
37

38. Foreground selection
 RM3412,
 RM140,
 RM493
Recombination selection
 RM1287, RM10843, RM10852,
RM562 and RM7075
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40. 40

41. 3. Marker-assisted pyramiding
 Widely used for combining multiple disease resistance
genes for specific races of a pathogen
 Pyramiding is extremely difficult to achieve using
conventional methods
 Important to develop ‘durable’ disease resistance
against different races of pathogen
41

42. Marker Assisted Gene Pyramiding
Assembling multiple desirable genes from multiple parents into single
genotype
(Hospital et al., 2004)
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43. 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
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44. A. Stepwise transfer;
B. Simultaneous transfer;
C. Simultaneous and stepwise transfer
44

45.  Blast, caused by the fungus Magnaporthae grisea is
one of the most serious diseases of rice (Oryza
sativaL.) worldwide
 Three major genes for blast resistance, Pi1, Piz-5
and Pita on chromosomes 11, 6, and 12, in three
NILs, C101LAC, C101A51 and C101PKT respectively.
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46. 46

47. 47

48. RG64 (SAP) and RG456:Piz-5
RZ536 :Pi1
RZ397 and RG869 for Pita
48

49.  The Xa21gene (resistance to bacterial blight), the
Bt fusion gene (for insect resistance) and the
chitinase gene-RC7 (for tolerance of sheath blight)
were combined in a single rice line by reciprocal
crossing of two transgenic homozygous IR72 lines.
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52. 52

53. 4. Early generation marker-assisted selection
MAS conducted at F2 or F3 stage
Very useful in self pollinated crops to fix alleles in their
homozygous state as early as possible
Allows breeders to focus attention on a lesser number of
high-priority lines in subsequent generations
 Linkage between the marker and QTL is not very tight,
 Disadvantage is the cost of genotyping a larger number
of plants.
53

54. F2
P2
F1
P1 x
large populations (e.g. 2000 plants)
ResistantSusceptible
MAS for 1 QTL – 75% elimination of (3/4) unwanted genotypes
MAS for 2 QTLs – 94% elimination of (15/16) unwanted genotypes
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55. Benefits: breeding program can be efficiently scaled down to focus
on fewer lines
Ribaut & Betran (1999)
SINGLE-LARGE SCALE MARKER-ASSISTED
SELECTION (SLS-MAS)
55

56. 5. Combined MAS approaches
In some cases, a combination of phenotypic screening
and MAS approach may be useful
1. To maximize genetic gain (when some QTLs have
remained unidentified)
2. Especially when large population sizes are used and trait
heritability is low
3. Low level of recombination between marker and QTL
4. To reduce population sizes for traits where marker
genotyping is cheaper or easier than phenotypic
screening
56

57. ‘Marker-directed’ phenotyping
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
Han et al (1997). 57

58. REASONS TO EXPLAIN THE LOW IMPACT
OF
MARKER-ASSISTED SELECTION
1.Still at the early stages of DNA marker technology
development
Even though SSR markers were developed in 1990’s
the availability is less
58

59. A significant gap between QTL discovery and their utilization in MAS is
depicted.
2. Marker-assisted selection results may not be
published
59

60. 3. Reliability and accuracy of QTL mapping
studies
 No. of replication used to generate phenotypic
data
 Population size
 Sampling bias
 Factors may affect the accuracy of a QTL mapping
60

61. 4. Insufficient linkage between marker and gene/QTL
5. Limited markers and limited polymorphism of
markers in breeding material
6. ‘Application gap’ between research laboratories
and plant breeding institutes
7. ‘Knowledge gap’ among molecular biologists, plant
breeders and other disciplines
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62. 8. Effects of genetic background
 QTLs identified in a particular mapping population
may not be effective in different backgrounds
(Liao et al.2001)
 Only one of four root length QTLs were effective
when transferred by backcrossing into a new rice
variety.
Steele et al. (2006)
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63. 9. Quantitative trait loci × environment interaction
 The magnitude of effect and even direction of QTLs
may vary depending on environmental conditions
Li et al.2003
10. High cost of marker-assisted selection
 The inheritance of the trait,
 The method of phenotypic evaluation,
 The cost of field and glasshouse trials and labour costs
 Large initial capital investments on purchase of
equipment, and regular expenses for maintenance.
63

64. Commercially available MAS varieties developed by public
institution
64

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66. 66

67. Examples of traits, for which marker assisted breeding is
being applied in rice
67

68. Research projects for the nutritional improvement
of varieties by marker assisted selection
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