Marker-Assisted Selection (MAS)/

Marker-Aided Selection (MAS)/
Marker-Assisted Breeding (MAB)

Gurbachan S. Miglani
Visiting Professor

School of Agricultural Biotechnology

Punjab Agricultural University
Ludhiana
What is Marker-Assisted selection?

An indirect selection process where a trait of

interest is selected based on a  marker
linked to a trait of interest

Assumption of MAS
The assumption is that the marker associates at
high frequency with the gene or quantitative trait
locus (QTL) of interest, due to genetic linkage
 Marker Types
Marker Type Description
Morphological Often detectable by eye, by simple visual inspection
Biochemical Protein can be extracted and observed; for
example, isozymes and storage proteins
Cytological Chromosomal banding produced by different stains
DNA-based/ A unique gene (DNA sequence), occurring in
Molecular  proximity to the gene or locus of interest, can be
identified by a range of molecular techniques

Morphological markers generally used for selection

Demerits of Morphological Markers

• Expression development specific

• Dominance
• Deleterious effects
• Pleiotropy
• Epistasis
• Rare polymorphism
 DNA-based or Molecular Markers
• Restriction Fragment Length Polymorphism (RFLP)
• Random Amplification of Polymorphic DNA (RAPD )
• Amplified Length Polymorphism (AFLP)
• DNA amplification fingerprinting (DAF)
• Sequenced Characterized Amplified Region Marker (SCAR)
• Simple sequence repeats (SSR) or microsatellites
• Single-nucleotide polymorphism (SNP)

In the marker aided selection, RFLP markers are widely used

for genetic improvement of crop plants for various
economic characters
 Pre-requisites for MAS
• Tight linkage between molecular marker
and gene of interest
• High heritability of the gene of interest
  Gene of Interest
DNA sequence that directly causes production of protein(s)
or RNA that produce a desired trait or phenotype

Marker
DNA sequence or the morphological or biochemical
markers produced due to that DNA are genetically
linked to the gene of interest
 An Ideal DNA-based Marker for MAS
An ideal marker should have the following properties:
• Easy recognition of all phenotypes from all different alleles
• Codomimant
• Low or null interaction among the markers
• Abundant in number
• Polymorphic
• Reliable
• Cost effective
 Reliability of DNA Markers
Markers should be tightly linked to target loci,
preferably less than 5 cM genetic distance

Use of flanking markers or intragenic markers greatly

increase the reliability of the markers to predict phenotype
Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans R Soc Lond B Biol Sci. 363 (1491): 557–572
Quantity and quality of DNA required
• Some marker techniques require large amounts
and high quality of DNA
• This adds to the cost of the procedures

Technical procedure for marker assay

• High-throughput simple and quick methods are
highly desirable

Cost
• Must be cost-effective
 Cost – Major Obstacle for Application of MAS

Estimates of costs (consumables and labour) for marker genotyping during MAS
Institute country crop species cost estimate (US$)
IRRI The Philippines rice 0.30, 1.00
University of Guelph Canada bean 2.74
CIMMYT Mexico maize 1.24–2.26
University of Adelaide Australia wheat 1.46
NSW Department of Agriculture Australia wheat 4.16
University of Kentucky, University of United States wheat and barley 0.50–5.00
Minnesota, University of Oregon, Michigan
State University, USDA-ARS

Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans R Soc Lond B Biol Sci. 363 (1491): 557–572
 Markers linked to the trait of interest are identified by QTL mapping
Single- ↓
step The same information is later used in the same population
↓
MAS Families are created by crossing number of parents
and QTL (in three-way or four way crosses)
↓
Mapping Pedigree structure is created
↓
Phenotyping and genotyping is done using molecular markers
↓
Map the possible location of QTL of interest
↓
This will identify markers and their favorable alleles
↓
The frequency of such alleles increased
↓
for breeding Estimate response to marker-assisted selection
typical plant ↓
populations Marker allele(s) with desirable effect will be further used
 Genotyping Techniques
• High-throughput genotyping techniques allow marker-aided
screening of many genotypes
• This helps breeders in shifting traditional breeding to marker-aided
selection
• One example of such automation is using DNA isolation robots,
capillary electrophoresis and pipetting robots
• One recent example of capillary
system is Applied Biosystems 3130
Genetic Analyzer.
• This is the latest generation of 4-
capillary electrophoresis
instruments for the low- to
ABI 3130 genetic analyzer
medium-throughput laboratories
 MAS for Backcross Breeding
A minimum of 5 to 6-backcross generations are required to
transfer a gene of interest from a donor to a recipient
↓
The recovery of the recurrent genotype can be accelerated
with the use of molecular markers
↓
If the F1 is heterozygous for the marker locus, individuals with the
recurrent parent allele(s) at the marker locus in first or subsequent
backcross generations will also carry a chromosome tagged by the marker
 Marker Development

Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans

R Soc Lond B Biol Sci. 363 (1491): 557–572
Steps in Marker-Assisted Selection (MAS)
• Selection of parents
• Development of breeding population
• Isolation of DNA from each plant
• Scoring RFLPs
• Correlation with morphological traits
 1. Selection of parents
• Parents with contrasting characters or divergent
origin should be chosen
• Select parents with distinct DNA
• This will help in identification of DNA of both the
parents and also their segments in F2 generation
2. Development of breeding population
• The selected parents are crossed to obtain F1 plants
• F1 plants between two pure-lines or inbred lines are
homogeneous
• F1 plants are heterozygous for all the RFLPs of the two
parents involved in the F1
• Generally 50-100 F2 plants are sufficient for the study
of segregation of RFLP markers
 3. Isolation of DNA from each plant
• DNA is isolated even from the seedlings and we need not wait for
flowering or seed development stage
• The DNA is isolated from each plant of F2 population
• The isolated DNA is digested with specific restriction enzyme to
obtain fragments of DNA
• The DNA fragments are separated by subjecting the digested DNA
to agarose gel electrophoresis
• The gel is stained with ethidium bromide
• The variation in DNA fragments can be viewed in the ultraviolet
light
• It is tedious to identify individual DNA fragment in such cases
 4. Scoring RFLPs
• The polymorphism in RFLPs between the parents and their
involvement in the recombinants in F2 population is
determined by using labelled DNA probes
• Generally 32P is used for radioactive labelling of DNA probe
• Now non-radioactive probe labelling techniques are also
available
• The probe will hybridize only with those DNA segments
which are complementary in nature
5. Correlation with morphological traits
• The DNA marker (e.g., RFLPs) is correlated with
morphological markers and the indirect selection through
molecular markers is confirmed
• Once the correlation of molecular markers is established
with morphological markers, MAS can be effectively used
for genetic improvement of various economic traits
 Advantages of MAS over Conventional
Phenotypic selection
• It may be simpler than phenotypic screening
o saves time, resources and effort

• Selection can be carried out at the seedling stage

o useful for traits that are expressed at later developmental stages

• Single plants can be selected based on their genotype

o homozygous and heterozygous plants can be distinguished

 These advantages accelerate the breeding process

 Many lines can be discarded after MAS early in a breeding scheme
 Permits more efficient use of glasshouse and/or field space
 Applicability of MAS
• MAS is applicable for genetic improvement of plants
as well as animals
• In plants, MAS is equally applicable in both self-
pollinated and cross-pollinated species

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
5. Combined marker-assisted selection
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
 2. Marker-assisted backcrossing (MAB)

• Backcrossing is most commonly used to incorporate one or

a few genes into an adapted or elite variety
• In most cases, the parent used for backcrossing has a large
number of desirable attributes but is deficient in only a few
characteristics
• The use of DNA markers in backcrossing greatly increases
the efficiency of selection
• Three general levels of MAB exist
 General Levels of MAB
Foreground selection Recombinant selection Background selection

Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans R Soc Lond B Biol Sci.
363 (1491): 557–572.
 Foreground selection (FS)

• Markers can be used in combination with or to replace

screening for the target gene or QTL
• FS may be particularly useful for traits that have laborious or
time-consuming phenotypic screening procedures
• FS can also be used to select for reproductive-stage traits in
the seedling stage, allowing the best plants to be identified for
backcrossing
• Recessive alleles can be selected, which is difficult to do using
conventional methods
 Recombination selection (RS)

• RS involves selecting BC progeny with the target gene and

recombination events between the target locus and linked
flanking markers
• RS reduces the size of the donor chromosome segment
containing the target locus
• 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, RS is usually performed
using at least two BC generations
 Background selection (BS)

• BS involves selecting BC progeny with the greatest

proportion of recurrent parent (RP) genome, using
markers that are unlinked to the target locus
• BS markers are used to select against the donor genome
• BS is extremely useful because the RP recovery can be
greatly accelerated
• It can be achieved by BC4, BC3 or even BC2
 Some Examples of MAB in Cereals
Foreground and background selection used

• Barley
• Maize
• Rice
• Wheat
Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans R Soc
Lond B Biol Sci. 363 (1491): 557–572
 3. Marker-assisted pyramiding

• The process of combining several genes together into a single

genotype
• DNA markers can greatly facilitate selection because DNA
marker assays are non-destructive and markers for multiple
specific genes can be tested using a single DNA sample
without phenotyping
• Pyramiding has been used for combining multiple disease
resistance genes (i.e., combining qualitative resistance genes
together into a single genotype)
• The combination of multiple genes (effective against specific
races of a pathogen) can provide durable (broad spectrum)
resistance
• MAS could be used to pyramid genes from multiple parents
Some Examples of Gene or QTL pyramiding in cereals

• Barley
• Rice
• Wheat

Collard, B.C.Y and D.J. Mackill. 2008. Philos Trans R Soc Lond B Biol Sci.
363 (1491): 557–572
 4. Early generation marker-assisted selection
• MAS is a great advantage in early generations because plants
with undesirable gene combinations can be eliminated
• When the linkage between the marker and the selected QTL is
not very tight, the greatest efficiency of MAS is in early
generations due to the increasing probability of recombination
between the marker and QTL
• The major disadvantage of applying MAS at early generations
is the cost of genotyping a larger number of plants
 Single Large-Scale MAS (SLS–MAS) Approach

• Proposed by  Ribaut and Betran (1999)

• A single-step MAS could be performed on F2 or F3 populations
derived from elite parents
• This approach used flanking markers (<5 cM, on both sides of a
target locus) for up to three QTLs in a single MAS step
 5. Combined marker-assisted selection

• Phenotypic screening can be strategically combined with MAS

• Combined MAS could be adopted when additional QTLs
controlling a trait remain unidentified or when a large number
of QTLs need to be manipulated
• Also referred to as ‘tandem selection’ or ‘stepwise selection’
• In wheat, MAS combined with phenotypic screening was
more effective than phenotypic screening alone for a major
QTL on chromosome 3BS for Fusarium head blight resistance
 Uses of Marker-Assisted Selection (MAS)
in Plant Breeding
• for transfer of male sterility and photoperiod insensitivity
into cultivated genotypes
• for improvement of quality characters in different crops
• for transfer of desirable transgene (such as Bt gene) from one
cultivar to another
• very effective in introgression of desirable genes from wild
into cultivated genotypes
• equally effective in genetic improvement of plants and
animals
• genetic improvement of tree species where fruiting takes very
long time
• wide application for genetic improvement of oligogenic as well
as compared to polygenic traits
Limitations of Molecular Breeding through MAS

• MAS only works for traits already present in a crop

• MAS cannot be used effectively to breed crops which have long
generation time (e.g., citrus)
• MAS cannot be used effectively with crops which are clonally
propagated because they are sterile or do not breed
• MAS is very costly as compared to phenotypic selection – the
costly items include equipment’s, consumables, infrastructure,
labour and DNA extraction process
• MAS requires sophisticated and well-equipped laboratory
Why Low Impact of Marker-Assisted Selection?
• Still at the early stages of DNA marker technology development
• Marker-assisted selection results may not be published
• Reliability and accuracy of quantitative trait loci mapping studies
• Insufficient linkage between marker and gene/QTL
• Limited markers and polymorphism of markers in breeding material
• Effects of genetic background
• Quantitative trait loci by environment effects
• High cost of marker-assisted selection
• ‘Application gap’ between research laboratories and plant breeding
institutes
• ‘Knowledge gap’ among molecular biologists, plant breeders and
other disciplines
 Adoption of MAS in Plant Breeding
Factors that will give rise to a much greater level of adoption of MAS:

• The extent to which DNA marker technology spreads to plant breeding

institutes
• Combining QTL mapping and MAS breeding
• MAS can be used to directly select for progeny that possess transgenes
via target gene selection
• SNPs within candidate genes could be extremely useful for ‘association
mapping’ and ultimately MAS
• New high-throughput methods for DNA extraction and new high-
throughput marker genotyping platforms developed
• The availability of large numbers of publicly available markers and the
parallel development of user-friendly databases for the storage of
marker and QTL data will encourage widespread use of MAS
 Realizing the Potential of MAS
It will be desirable to have
• a greater level of integration among conventional breeding,
QTL mapping/validation and MAS
• careful planning and execution of QTL mapping studies and an
emphasis on validating results prior to MAS
• optimization of methods used in MAS such as DNA extraction
and marker genotyping, especially in terms of cost reduction
and efficiency, and
• efficient systems for data storage
• Advantages of MAS over conventional breeding need to be fully
exploited by
o use of markers for the selection of parents in breeding
programmes,
o continued use of MAS for high-priority traits that are difficult,
time consuming or expensive to measure
o using markers to minimize linkage drag via recombinant selection
o screening of multiple traits per line, especially populations
derived from multiple F1s for pyramiding
o exploiting the ability to rapidly eliminate unsuitable lines after
early generation selection or tandem selection in breeding
programmes
o exploiting the time savings for line development
o can be used for improvement of both oligogenic and polygenic
traits
o leads to development of non-transgenic genotypes or cultivars
 Achievements of MAS
Rice • For bacterial blight resistance four genes (Xa4, Xa5, Xa13 and Xa21) have been
pyramided using STS (sequence tagged site) markers.
• Two bacterial blight resistant varieties of rice – Angke and Conde – have been
released through MAS
• For blast resistance, three genes (Pil, Piz5 and Pita) have been pyramided in a
susceptible rice variety Co39 using RFLP and PCR-based markers
Maize • Normal lines have been converted into quality protein maize (QPM) lines through
MAS using opaque 2 recessive allele at CIMMYT, Mexico)
• Three SSR markers (Umc 1066, Phi 057 and Phi 112) present within opaque 2 gene
have been used for this purpose
• The MAS used for conversion of normal maize lines into QPM is simple, rapid and
accurate
Soybean • Nematode resistant lines have been developed through MAS using SSR marker Sat
309
Tomato • More than 40 genes that confer resistance to major classes of tomato pathogens
have been mapped, cloned, and/or sequenced
• These maps have allowed for “pyramiding” resistance genes in tomato through
MAS, where several resistance genes can be engineered into one genotype
• Tomato breeding through MAS has resulted in varieties with resistance or
tolerance to one or more specific pathogens
 Advantages of MAS
Accuracy • Molecular markers are not affected by environmental conditions
• MAS is very effective even with the characters having low
heritability
Rapid Method • Using MAS, It takes only 3-5 years for developing a new cultivar
against 10-15 years taken by the conventional method of breeding
Non-transgenic • MAS leads to development of non-transgenic cultivars which are
Product acceptable to everybody
Identification • MAS is equally effective for the genetic improvement of recessive
of Recessive characters as it permits identification of recessive alleles even in
Alleles heterozygous condition
Early Detection • MAS permits early detection of traits that are expressed late in the
of Traits life of plant
Screening of • MAS permits screening traits such as root morphology and resistance
Difficult to and abiotic stresses
Traits
Gene • MAS provides an effective and efficient breeding tool for detecting,
Pyramiding tracking, retaining, combining and pyramiding genes for disease
resistance
Small • MAS requires only a small amount of plant tissue for DNA testing
Sample for • MAS can be carried out with small breeding populations
Testing • MAS can be applied at any stage of plant growth
Permits QTL • MAS permits mapping or tagging of quantitative trait loci (QTL)
Mapping
Highly • MAS is based on DNA fingerprinting technique
Reproducible • The results of DNA fingerprinting pattern are highly reliable and
reproducible

Mohler, V. & Singrun, C. 2004. In Biotechnology in Agriculture and Forestry, Vol.

55: Molecular marker systems (eds H. Lorz & G. Wenzel), pp. 305–317. Berlin,
Germany: Springer