The document discusses marker-assisted selection (MAS) in crop breeding, which utilizes DNA markers linked to target traits to streamline the breeding process by reducing time and costs associated with phenotypic screening. It details various types of markers, essential properties of ideal markers, and prerequisites for efficient MAS programs, as well as the advantages and potential benefits, including more accurate genotype selection. Additionally, various methodologies within MAS, such as marker-assisted backcrossing and recurrent selection, are outlined to enhance genetic gains in plant breeding.

1. MARKER ASSISTED SELECTION IN
CROP BREEDING
Presented By:– Pawan Kumar
ROLL. NO. – 143G03
Deptt:- Plant Breeding & Genetics
Submitted To:-
Dr. A. K. Saxena

2. MAS
 Marker assisted selection
The use of DNA markers that are tightly-linked to target
loci as a substitute for or to assist phenotypic screening
DNA markers can reliably predict phenotype
Assumption

3. Breeding for specific traits in plants is expensive and time
consuming
The progeny often need to reach maturity before a
determination of the success of the cross can be made
The greater the complexity of the trait, the more time and
effort needed to achieve a desirable result
The goal to MAS is to reduce the time needed to determine if
the progeny have trait
The second goal is to reduce costs associated with screening
for traits
If we can detect the distinguishing trait at the DNA level we
can identify positive selection very early.

4. Gene vs. Markers
 The gene of interest directly causes production of protein(s) or
RNA that produce a desired trait or phenotype.
 Markers (a DNA sequence or the morphological or biochemical
markers produced due to that DNA) are genetically linked to the
gene of interest.
 The gene of interest and the marker tend to move together during
segregation of gametes due to their proximity on the same
chromosome and concomitant reduction
in recombination (chromosome crossover events) between the
marker and gene of interest.
 If the gene of interest is not known, markers linked to the
gene of interest can still be used to select for individuals with
desirable alleles of the gene of interest.
 The term 'perfect marker' is sometimes used when tests are
performed to detect a SNP or other DNA polymorphism in the
gene of interest,

5. Marker types
Morphological markers: markers are often detectable by eye, by
simple visual inspection. leaf sheath coloration, height, grain color,
aroma of rice etc.
Biochemical Markers: A protein that can be extracted and
observed; for example, isozymes and storage proteins.
Cytological Markers: The chromosomal banding produced by
different strains; for example, G banding.
DNA based or Molecular Markers: A unique gene (DNA
sequence), occurring in proximity to the gene or locus of interest,
can be identified by a range of molecular techniques such
as RFLP, RAPD, AFLP, DAF, SCAR, microsatellite, or single-
nucleotide polymorphism (SNP) detection.

6. Important properties of ideal
markers for MAS
 Easy recognition of all possible phenotypes (homo-
and heterozygotes) from all different alleles
 Demonstrates measurable differences in expression between trait
types or gene of interest alleles, early in the development of the
organism
 Testing for the marker does not have variable success depending
on the allele at the marker locus or the allele at the target locus (the
gene of interest that determines the trait of interest).
 Low or null interaction among the markers allowing the use of
many at the same time in a segregating population
 Abundant in number
 Polymorphic.

7. Prerequisites for an efficient
marker-assisted breeding program
 Appropriate marker system and reliable markers: 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 markers should have following
attributes:
 Ease and low-cost of use and analysis;
 Small amount of DNA required;
 Co-dominance;
 Repeatability/reproducibility of results;
 High levels of polymorphism; and
 Occurrence and even distribution genome wide

8. F2
P2
F1
P1 x
large populations consisting of thousands of plants
PHENOTYPIC SELECTION
Field trials
Glasshouse trials
DonorRecipient
CONVENTIONAL PLANT BREEDING
Salinity screening in
phytotron
Bacterial blight screening Phosphorus deficiency plot

9. 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

10. Activities of marker-assisted
breedingMarker-assisted breeding involves the following activities provided the
prerequisites are well equipped or available:
 Planting the breeding populations with potential segregation for traits
of interest or polymorphism for the markers used.
 Sampling plant tissues, usually at early stages of growth, e.g. emergence
to young seedling stage.
 Extracting DNA from tissue sample of each individual or family in the
populations, and preparing DNA samples for PCR and marker
screening.
 Running PCR or other amplifying operation for the molecular markers
associated with or linked to the trait of interest.
 Separating and scoring PCR/amplified products, by means of
appropriate separation and detection techniques, e.g. PAGE, AGE, etc.
 Identifying individuals/families carrying the desired marker alleles.
 Selecting the best individuals/families with both desired marker alleles
for target traits and desirable performance/phenotypes of other traits,
by jointly using marker results and other selection criteria.

11. The situations favorable for MAS include
 The selected character is expressed late in plant
development, like fruit and flower features or adult
characters with a juvenile period
 The target gene is recessive
 Special conditions are required in order to invoke expression
of the target gene(s), as in the case of breeding for disease
and pest resistance or the expression of target genes is
highly variable with the environments.
 The phenotype of a trait is conditioned by two or more
unlinked genes.

12. 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

13. 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

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

15. Marker assisted breeding schemes
1. Marker-assisted backcrossing
2. Marker-assisted recurrent selection (MARS)
3. Gene Pyramiding
4. Early generation selection
5. ‘Combined’ approaches

16. Marker-assisted
backcrossing
The general procedure of MABC is as follow
 Select parents and make the cross, one parent is recurrent parent
(RP), and the other one used as donor parent (DP)
 Plant F1 population and detect the presence of the marker allele(s) at
early stages of growth to eliminate false hybrids, and cross the true
F1 plants back to the RP.
 Plant BCF1 population, screen individuals for the marker(s) at early
growth stages, and cross the individuals carrying the desired marker
allele(s) (in heterozygous status) back to the RP.
 Plant the final backcrossing population (e.g. BC4F1), and screen
individual plants with the marker(s) for the target trait. Have the
individuals with required marker allele(s) selfed and harvest them.
 Plant the progenies of backcrossing-selfing (e.g. BC4F2), detect the
markers and harvest individuals carrying homozygous DP marker
allele(s) of target trait for further evaluation and release.

17. Marker-assisted backcrossing (MAB)
1 2 3 4
Target
locus
1 2 3 4
RECOMBINANT
SELECTION
1 2 3 4
BACKGROUND
SELECTION
TARGET LOCUS
SELECTION
FOREGROUND
SELECTION BACKGROUND SELECTION

18. Marker-assisted recurrent selection (MARS)
 The long selection cycles impose restrictions on the
practicability of recurrent selection method of breeding.
 In continuous nursery programs pre flowering genotypic
information is used for marker assisted selection and
controlled pollination.
 It is possible today to define an ideal genotype as a pattern
of QTLs, all QTLs carrying favorable alleles from various
parents.
 It is likely that through such a MARS breeding scheme
higher genetic gain will be achieved than through MABC.

19. Gene 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

20. 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

21. 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

22. 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

23. 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

24. Combined approaches
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

25. ‘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