Marker assisted selection is the breeding strategy in which selection for a gene is based on molecular markers closely linked to the gene of interest rather than the gene itself, and the markers are used to monitor the incorporation of the desirable allele from the donor source. Selection of a genotype carrying desirable gene via linked marker (s) is called Marker Assisted Selection. MAS can be applied to possible to use this kind of information. The prerequisites for the classical procedure of MAS are the tight linkage between molecular marker and gene of interest and high heritability of the gene of interest. It is noteworthy that the “quality” and the number of markers have a major impact on the success of MAS. The quality of markers relates to their characteristics and to the cost and the efficiency of the genotyping process. The number of markers affects the reliability of the linkage between them and the gene(s). In other words, screening a large number of markers has the potential to identify close and reliable linkage between the marker and the gene of interest. MAS has greater potential for efficient gene pyramiding combining several important genes in one cultivar. MAS is gaining considerable importance as it can improve the efficiency of plant breeding through precise transfer of genomic regions of interest and acceleration of the recovery of the recurrent parent genome. Marker-assisted selection is gaining considerable importance as it would improve the efficiency of plant breeding through precise transfer of genomic regions of interest (foreground selection) and accelerating the recovery of the recurrent parent genome (background selection). The use of MAS in crop improvement will not only reduce the cost of developing new varieties but will also increase the precision and efficiency of selection in the breeding program as well as lessen the number of years required to come up with a new crop variety.

1. Marker-Assisted Selection
and Its Role In Crop Improvement
Khushbu (A-2018-40-015)
PhD. Student
(Department of Genetics and Plant Breeding)
Date : 29-11-19

2. CONVENTIONAL PLANT BREEDING:
Conventional plant breeding is the
development or improvement of cultivars
using conservative tools for manipulating plant
genome within the natural genetic boundaries
of the species.

3. Marker-assisted selection (MAS) refers to use of
molecular markers to assist phenotypic selection
in crop improvement.
Based on the concept: It is possible to infer
presence of a gene from the presence of a marker
which is tightly linked to a gene of interest.
MARKER ASSISTED SELECTION

4. CONVENTIONAL METHOD MARKER ASSISTED SELECTION

5. • MAS can be performed on seedlings.
• Not affected by environmental conditions.
• Recessive allele detection.
• Combining multiple genes simultaneously.
• Cheaper and faster depending upon trait.
• Testing for specific traits where phenotypic
evaluation is difficult.
• Minimize linkage drag.
WHY MARKER ASSISTED SELECTION?

6. Population development
Parental selection and hybridization
QTL mapping
Linkage map construction/ phenotypic evaluation of trait/ QTL analysis
Marker validation
Testing of marker in important breeding material
Marker assisted selection
MARKER DEVELOPMENT PIPELINE

7. • Any genetic element of organism whether
phenotypic level or molecular level which can
help to identify the desirable traits/ characters
• A chromosomal landmark or allele that allows
for tracing of a specific region of DNA.
MARKERS
(Semagn et al. 2006)

8. MARKER
FOUR TYPES
MORPHOLOGICAL BIOCHEMICAL CYTOLOGICAL MOLECULAR

9. • Usually these are visually characterized
phenotypic characters such as flower colour,
seed shape and growth habit.
• These traits are scorable by the naked eye, also
termed as naked eye polymorphisms.
• Scoring of these markers is simple, rapid and
inexpensive and often can be scored even from
preserved specimens (Stussey 1990).
Morphological Markers

10. Biochemical based markers
 Isozyme - a molecular marker system based
on the staining of proteins with identical
function, but different electrophoretic
mobilities.
 Enzyme polymorphisms have been used
successfully to identify cultivars in various fruit
species.

11. • The DNA based makers differentiate the
organisms at DNA level and are inherited in
simple Mendelian fashion.
• This is particularly important for genetic
resource management, as well as for the rational
use of genetic resources in selection programs.
• DNA markers can detect differences in genetic
information carried by two or more individuals.
DNA based markers

12. Limitations of
morphological markers
Merits of molecular
markers
Scored on whole plant that too
on specific developmental
stages.
Scoring performed on seedling
stage.
Highly influenced by the
environmental factors.
Not affected by environmental
conditions.
Maintenance of suitable genetic
stocks expressing the various
marker traits would be
necessary.
Result are reproducible.

13. DNA markers broadly divided into three classes
based on their method of detection:
• Hybridisation based
• Polymerase chain reaction-based
• DNA–sequence based
DNA based markers

14. Advantages and disadvantages of commonly-used DNA markers
Molecular
marker
Codominant
or Dominant
Advantages Disadvantages References
RFLP Codominant • Robust
• Reliable
• Transferable
across
populations
• Time-
consuming,
laborius and
expensive
• Large amounts
of DNA required
• Limited
polymorphisms
Beckmann &
Soller
(1986),
Kochert
(1994),
Tanksley et
al. (1989)
RAPD Dominant • Quick and simple
• Inexpensive
• Multiple loci
from a single
primer possible
• Small amounts of
DNA required
• Problems with
reproducibility
• Generally not
transferable
Penner
(1996),
Welsh &
McClelland
(1990),
Williams et
al. (1990)

15. Advantages and disadvantages of commonly-used DNA markers
Molecular
marker
Codominant
or Dominant
Advantages Disadvantages Refrences
SSR Codominant • Technically
simple
• Robust and
reliable
• Transferable
between
populations
• Large amount
of time &
labour required
for production
of primers
• Require
polyacrylamide
electrophoresis
McCouch et al.
(1997), Powell et
al. (1996),
Taramino and
Tingey (1996)
AFLP Dominant • Multiple loci
• Highly
polymorphic
• Large amount
of DNA
required
• Complicated
methodology
Vos et al. (1995)

16. Advantages and disadvantages of commonly-used DNA markers
Molecular
marker
Codominant or
Dominant
Advantages Disadvantages
SNP Codominant • Cost effective
• High reproducibility,
• Widely distributed in
genome
• High
developmental
cost
SCARS Codominant • Simpler patterns than
RAPDs (locus
specific)
• Robust assay
• Sequence
information
needed
• Require efforts
and expense in
designing of
primers

17. SELECTION OF
MARKER
TECHNOLOGY
RESEARCH
PROBLEM
NO. OF LOCI OR
ALLELES
EXPERTISE
REQUIRED
DISCRIMINATION
LEVEL
SPEED
MODE OF
INHERITANCE
QUALITY OF
DNA
SELECTION OF MARKER TECHNOLOGY

18. APPLICATION OF MAS
• MARKER ASSISTED BACKCROSSING
• GENE PYRAMIDING
• MARKER BASED RECURRENT SELECTION
• MARKER-ASSISTED EVALUATION OF BREEDING
MATERIAL
• EARLY GENERATION MARKER ASSISTED SELECTION
(Collard and Mackill, 2016)

19. 1 2 3 4
Target locus
TARGET LOCUS
SELECTION
RECOMBINANT
SELECTION
1 2 3 4
1 2 3 4
BACKGROUND
SELECTION
MARKER ASSISTED BACKCROSSING

20. • Marker should
be tightly linked
with the target
gene
• Useful for traits
that are difficult
to evaluate
1ST LEVEL OF SELECTION:
FOREGROUND SELECTION

21. 2ND LEVEL OF SELECTION:
RECOMBINANT SELECTION
i) Use flanking markers to
select recombinants
between the target locus
and flanking marker
ii) Linkage drag is minimized
iii) Require large population
sizes – depends on
distance of flanking
markers from target
locus)

22. 3RD LEVEL OF SELECTION
BACKGROUND SELECTION
i) Accelerates the
recovery of the
recurrent parent
genome
ii) With conventional
backcrossing, it takes a
minimum of five to six
generations to recover
the recurrent parent.
iii) Savings of 2, 3 or even
4 backcross
generations may be
possible
1 2 3 4
BACKGROUND SELECTION

23. STEP TOWARDS PRODUCTIVITY BREEDING
TRIGUNA VARIETY
SUSCEPTIBLE TO
BB
INTROGRESSED
Xa21, Xa13, Xa5
FOREGROUND
SELECTION FOR 3
GENES AT EACH
GENERATION
BACKGROUND
SELECTION FOR
TRIGUNA
GENOME
BC3F2
UPTO BC3F8
PLANTS WITH DISEASE
RESISTANCE GENES+ HIGH
YIELD
(Sundaram et al 2014)

24. Marker-assisted backcross breeding scheme adapted for the introgression of crtRB1 gene in
to elite parent (V335 and V345) of the maize hybrid Vivek Hybrid-27).
Muthusamy et al. (2014) Development of β-Carotene Rich Maize Hybrids through Marker-Assisted Introgression of β-
carotene hydroxylase Allele. PLOS ONE 9(12): e113583. https://doi.org/10.1371/journal.pone.0113583
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0113583

25. MARKER ASSISTED GENE PYRAMIDING
• Pyramiding is the process of simultaneously
combining multiple genes/QTLs together into a
single genotype.
• Most widespread application for pyramiding has
been for combining multiple disease resistance
genes in order to develop durable disease
resistance.

26. MARKER ASSISTED GENE PYRAMIDING

27. PCR based molecular markers are used in a backcross-breeding programme to introgress
three major BB resistance genes
Xa21,
Xa13,
Xa5
into Samba Mahsuri from a donor line (SS1113) in which all the three genes are present in
a homozygous condition.
Samba Mahsuri is a medium slender grain
indica rice variety that is very popular with
farmers and consumers across India because
of its high yield and excellent cooking quality.
However, the variety is susceptible to several
diseases and pests, including bacterial blight
(BB).
(Sundaram et al 2007)

28. MARKER ASSISTED GENE PYRAMIDING
EXAMPLE FROM PAU
• Xa13 – RG136
• Xa5– RG 556
• Xa21 – Pta248
3 GENES
PYRAMIDED
• MARKER ASSISTED
BACKCROSSING
PR 106 GENETIC
BACKGROUND • 17 FROM PUNJAB
• 6 FROM PHILLIPINES
• EVALUATED AT 31
SITES IN PUNJAB
(COMM. FIELDS)
EVALUATION WITH
XOO ISOLATES

29. PYRAMIDING OF TRANSGENES
LINES 910,912
CONTAINS ALL
NINE GENES
(BC6)
MAIZE A188
ANTI APOPTOSIS
GENES
(Iap, p35)
DEFENCE
RESPONSE GENES
(Chi, Glu, AceAMP1,
Tlp, Rs-AFP2,
ZmPROPEP1, Pti 4)

30. MARKER ASSISTED RECURRENT SELECTION
Recurrent selection scheme using
molecular markers for the identification
and selection of multiple genomic regions
involved in the expression of complex
traits to assemble the best performing
genotype within a single or a cross related
populations.
(Gokidi et al. 2016)

31. MARKER ASSISTED RECURRENT SELECTION

32. i) Cultivar identity/assessment of ‘purity’
ii) Assessment of genetic diversity and
parental selection
iii) Study of heterosis
iv) Identification of genomic regions under
selection
MARKER-ASSISTED EVALUATION OF
BREEDING MATERIAL

33. • MAS conducted at F2 or F3 stage.
• Undesirable gene combinations can be
eliminated.
• Advantageous for later stages of breeding
program because resources can be used to
focus on fewer lines.
• Plants with desirable genes/QTLs are
selected and alleles can be fixed in the
homozygous state.
EARLY GENERATION MARKER-ASSISTED
SELECTION

34. BIOINFORMATICS IN MAS
• Data grows exponentially.
• Demand for tools and methods in data management,
visualisation, integration, analysis, modelling and
prediction.
• Unfamiliarity to bioinformatics may lead to biased
interpretation of information.
Examples:
Primer designing softwares: Primer-Blast, Primer3, Primer3Plus etc.
Linkage analysis: MapMaker, PLABQTL, INTERQTL, QGene etc.

35. 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

36. 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

37. CROP VARIETY TRAIT MAS
MAIZE PUSA VIVEK QPM9 HIGH LYSINE+TRYPTOPHAN+
PRO-VIT A
+
PUSA HM4 HIGH LYSINE+ TRYPTOPHAN +
WHEAT WB02 HIGH Zn+ HIGH Fe +
PATWIN RUST RESISTANT +
BAJRA HHB229 HIGH Zn+ HIGH Fe +
BRASSICA PUSA DOUBLE ZERO
MUSTARD 31
ERUCIC ACID <2%,
GLUCOSINOLATES <30ppm
+
CAULIFLOWER PUSA BETA KESARI 1 BETA CAROTENE +
VARIETIES THROUGH MAS

38. Breeding line Year Designation Country
IR05F102 (Swarna) 2009 Improved Swarna India
2009 INPARA-5 Indonesia
2010 BRRI dhan-51 Bangladesh
2011 Swarna-Sub1 Nepal
2011 Yemyoke Khan Myanmar
IR07F102 (IR64) 2009 NSIC Rc194 Philippines
2009 INPARA-4 Indonesia
IR07F290 (BR11) 2010 BRRI dhan-52 Bangladesh
IR09F436 (Ciherang) 2011 Ciherang-Sub1 Indonesia
2013 Bina dhan 11 Bangladesh
IR07F101 2012 S. Mahsuri-Sub1 India
2011 S. Mahsuri-Sub1 Nepal
2013 Bina dhan 12 Bangladesh
MAS PRODUCTS - Sub1 VARIETIES
RELEASED IN ASIA

39. SUCCESS STORIES
CROP VARIETY YEAR TRAIT GENES REMARKS
RICE Punjab
Basmati 3
2013 BB resistant and
improved version of
Basmati 386
Xa13, Xa21 Product of MAS
technology developed
by PAU
PAU 201 Grain color and BB
resistance
Rc7, Xa21 Under evaluation for
identifying lines to be
released as variety
WHEAT Unnat
PBW 343
2017 Rust resistance Yr 17/ Lr37/Sr38
; Yr40/Lr57
First wheat variety
through MABB
Unnat PBW
550
2017 Stripe rust resistance Yr15 Released at national
level
PBW 1 Zn 2017 High grain Zn +Fe
from wild diploid
wheats and cultivated
wheats
incorporated
several novel
alleles for grain
Zn
30–40% higher grain Zn
than local checks
COTTON PAU Bt 1 Resistance against
American, spotted and
pink bollworms
Cry1Ac First Bt cotton variety
developed by public
sector

40. WRAP - UP
• Use MAS where conventional selection is
costly or ineffective
• MAS should be explored to select
recombinants for productivity breeding
• MAS should focus on pyramiding
genes/QTLs having different mechanisms
of tolerance in breeding stress tolerant
cultivars.
• Efforts should also be made to exploit
minor effect QTLs
• Utilize bioinformatics tools in
management of exponentially growing
data.