Marker Types,Prerequisites for efficient marker-assisted breeding programmes,Advantages of MAS,Limitations of MAS ,Marker Assisted Breeding Schemes,• 1. Marker- assisted backcrossing,2. Marker- Assisted evaluation of breeding material,3 Gene pyramiding,4. Early generation selection ,Combined approaches,MAB: I level of Selection – FOREGROUND SELECTION,Second level of selection: Recombinant Selection,MAB: III Level of Selection BACKGROUND SELECTION,

1. Marker Assisted Selection and its application in
Plant breeding
Submitted to-
Dr. P.T.Patel/Mr. Kiran Kugashiya
Asso. Professor/Asstt. Professor
Seed Technology unit, SDAU.
Submitted by-
Hemantkumar S. Sonawane
Reg. No. 04-AGRPH-01857-2018 Ph. D.II yr
Department of Genetics and Plant Breeding
C. P. College of Agriculture, SDAU.
Course Name: GP 602:Advanced Biometrical and Quantitative Techniques

2. Marker Assisted Selection (MAS)
• Marker assisted selection (MAS) is indirect selection for a
gene /QTL based on molecular markers closely linked to the
gene /QTL
• A tool that can help plant breeders to select more efficiently
for desirable crop traits
• Molecular markers can also be used for negative selection
for elimination of undesirable genes, from segregating
population

3. Marker Assisted Selection
• Breeding for specific trait in plants is expensive & time consuming
• The first goal of MAS is to reduce the time needed to determine if
the progeny have the trait.
• The second goal is to reduce cost associated with screening for
traits
• If we can detect the distinguishing trait at the DNA level we can
identify positive selection very early.

4. What is Marker?
Marker is flag
(protein/DNA/morphology)
that is associated with a
certain trait of an organism
Morphological
Biochemical
Molecular
Cytological
Types of
Markers

5. Marker types
• Morphological markers : markers are often detected by eye , by
simple visual inspection. Example Leaf sheath coloration , height,
grain color, aroma of rice etc.
• Biochemical markers : A protein that can be extracted and observed .
eg. Isozymes & storage protein
• Cytological markers : The chromosomal banding produced by
different stains, for example G banding
• DNA based or Molecular markers : A unique gene( DNA),
occurring in proximity to the gene or locus of interest, can be identified
by a range of molecular techniques such as RFLP, RAPD, SCAR,
microsatellite or SNP detection

6. TYPES OF MOLECULER MARKERS
1. Morphological
2. Biochemical
 Classic Marker  DNA Marker
6
DNA Marker
A. CODOMINANT MARKERS
e.g. SSR , RFLP , Allozymes
B. DOMINANT MARKERS
e.g. RAPD , AFLP , SCAR
RFLP - Restriction Fragment Length Polymorphism
RAPD - Randomly Amplified Polymorphic DNA
AFLP - Amplification Fragment Length
Polymorphism
VNTR – Variable Number Tandem Repeat
SSR - Simple Sequence Repeats (microsatellites)
SNP - Single Nucleotide Polymorphism
SCAR - Sequence Characterized Amplified Regions
OP - Oligonucleotide Polymorphism
ASAP – Allele Specific Associated Primers
ISTR – Inverse Sequence-tagged Repeats
IRAP – Inter-retrotransposon Amplified
Polymorphism
TWO TYPES OF
MARKER
1. Non PCR
based
2. PCR based

8. Prerequisites for efficient marker-assisted breeding
programmes
• Appropriate marker system and reliable marker: For a plant species or crop,
a suitable marker system and reliable markers available are critically important to
initiate a marker- assisted breeding program. Suitable marker should have
following attributes :
• Ease and low –cost of use and analysis
• Small amount of DNA required
• Co-Dominance
• Repeatability/reproducibility of results
• High level of polymorphism
• Occurrence and even distribution genome wide

13. 13
Limitations of MAS
MAS is a costly method
 It requires well equipped laboratory
MAS requires well trained manpower for handling of sophisticated
equipments
The detection of various linked DNA markers (AFLP, RFLP, RAPD,
SSR, SNP etc.) is a difficult, laborious and time consuming task.
 health hazards

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

16. Marker Assisted Breeding Schemes
• 1. Marker- assisted backcrossing
• 2. Marker- Assisted evaluation of breeding material
• 3 Gene pyramiding
• 4. Early generation selection
• 5. Combined approaches

17. 1. Marker Assisted Backcrossing ( MAB)
• MAB is the simplest
form of MAS, in which
the goal is to incorporate
a major gene from an
agronomically inferior
source (the donor
parent) into an elite
cultivar or breeding line
(the recurrent parent).

18. (Marker Assisted Backcrossing)
NIL
P1 (RP) × P2 ( DP)
F1 P1×
BC1F1 P1×
BC2F1
BC2F2 BC3F1
Foreground selection for
Homozygous target gene and
Background selection for
Recurrent parent
Foreground selection for
target gene and
Background selection
for Recurrent parent
BC2F3
P1

19. • MAB has several advantages over conventional breeding.
Effective selection for target loci
Minimizes linkage drag
Accelerate recovery of recurrent parents
1. Marker Assisted Backcrossing ( MAB)

20. • In this level,markers can be used in combination
with target gene or QTL. This is referred as
‘foreground selection’ .
• This may be particularly useful for traits that have
laborious or time-consuming phenotypic screening
procedures.
• It can also be used to select for reproductive-stage
traits in the seedling stage, allowing the best plants
to be identified for backcrossing.
• Furthermore, recessive alleles can be selected,
which is difficult to do using conventional methods.
MAB: I level of Selection – FOREGROUND SELECTION

22. • Many undesirable genes that negatively affect crop performance may be
linked to the target gene from the donor parent—this is referred to as
‘linkage drag’.

24. • The second level involves selecting BC
progeny with the target gene and
recombination events between the target
locus and linked flanking markers termed as
‘recombinant selection’.
• The purpose of recombinant selection is to
reduce the size of the donor chromosome
segment containing the target locus
MAB:lI nd Level of selection
RECOMBINANT SELECTION

25. • Using conventional breeding methods, the donor segment can
remain very large even with many BC generations i.e. more
than 10.
• By using markers that flank a target gene (e.g. less than 5 cM
on either side), linkage drag can be minimized.
• Since double recombination events occurring on both sides of
a target locus are extremely rare recombinant selection is
usually performed using at least two BC generations

26. • The third level of MAB involves selecting BC
progeny with the greatest proportion of
recurrent parent (RP) genome, using markers
that are unlinked to the target locus we refer to
this as ‘background selection’.
• Background selection refers to the use of tightly
linked flanking markers for recombinant
selection and unlinked markers to select for the
RP.
MAB: III Level of Selection
BACKGROUND SELECTION

27. • With conventional backcrossing, it takes a minimum of 6 BC
generations to recover the RP and there may still be several
donor chromosome fragments unlinked to the target gene.
• The use of background selection during MAB to accelerate the
development of an RP with an additional (or a few) genes has
been referred to as ‘complete line conversion’

28. P1 P2 1 2 3 4 5 6 7 8 9 10
Background selection for recurrent parent
P1 P2 1 2 3 4 5 6 7 8 9 10

30. Examples of Marker-assisted Backcrossing in cereals

31. 2. Marker Assisted Evaluation of Breeding material
• Assessment of purity
• Assessment of genetic diversity and parental selection
• Study of heterosis e.g. Maize & sorghum
• Identification of genomic region under selection for QTL mapping &
development of variety through MAS

32. 3. Gene Pyramiding
• Gene Pyramiding is the process of combining several genes together
into a single genotype
• 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

33. 33
Gene Pyramiding
Process of combining several genes usually from 2 different parents, together into a single genotype
Breeding plan
×
F1
Gene A + B
MAS
Select F2 plants that have
Gene A and Gene B
Genotypes
P1 : AAbb × P2 : aaBB
P2P1
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
(Hittalmani & Liu et al., 2000)

34. Examples of gene or QTL pyramiding in cereals

35. 4. Early generation MAS
• MAS conducted in 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

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

38. References
• Collard B.C.Y. and Mackill D.J.2007 Marker assisted selection: An
approach for precision plant breeding in the twenty first century, Phil.
Trans. R. Soc. B , 363, 557–572
• Hittalmani, et al.,2000 Fine mapping and DNA marker-assisted
pyramiding of the three major genes for blast resistance in rice, Theor
Appl Genet ,100:1121–1128
• Liu et al 2000, Molecular marker facilitated pyramiding of different genes
for powdery mildew resistance in wheat, Plant Breeding 119,21-24
• Ribaut J.M and Hoisington D,1998 Markrt assisted selection: New tools
and stategies, Trends plant science 3, 336-339
• Singh B.D.2018 Plant breeding :principles and methods, Kalyani
publication, New Delhi :748-758

39. Thank You