Chapter 12 Molecular Markers

Chapter 12: Molecular Markers
Their Application in Crop

Improvement
Anon Chaulagain
Department of Biotechnology, HWIC
1
Marker
• Marker is an allelic difference or variation at a given locus in the
DNA that can be observed at morphological, biochemical or
molecular level
• Molecular marker are based on naturally occurring changes or

polymorphism in DNA sequence (deletion, substitution, addition,
tandem repeat or duplication)
• All molecular markers occupy specific genomic positions within

the chromosome known as “loci”
• Markers located in close proximity to desirable genes (tightly

linked) are known as “gene tags”
• Agriculturally important traits are governed by many genes

“Polygenic” (Quantitative traits)
• Regions in genome containing genes associated with a particular
quantitative trait are known as Quantitative Trait Loci (QTL)
Molecular markers are used because:
 they are selectively neutral as they are present in the non

coding region of the genome
 Makers are co-segregating with the trait of interest
 They follow exactly the Mendelian pattern of inheritance
 They are free from epistatic interaction or pleiotropic effect

Attributes of a perfect molecular marker
 Polymorphic
 Co-dominant
 Reproducible
 Robust
 Cost effective
 Easy to use
 High throughput
 Closely linked to the trait of interest

Level of analysis of markers
1. Morphological markers
• Are botanical descriptors of plant which are visually or
phenotypically characterized
• Also, known as DUS descriptors or universal markers (D -
Distinctiveness; U - Uniformity; S – Stability)
• For e.g. seed colour, seed shape, seed size, flower colour, growth
habits, plant pigmentation
2. Biochemical markers
• Biochemical markers are of two types:
 Protein based
 Enzyme based: Isozyme
• First true biochemical marker was an allelic variant of enzyme

pyruvate dehydrogenase
• Isoforms can be resolved by gel electrophoresis based on their

size, shape and amino acid differences
• Can be easily assayed and detected
• They are low in polymorphism as compared to DNA markers

3. DNA based markers
On the basis of ability to discriminate between same or different
species they are:
1. Co-dominant: discriminate between homo and heterozygotes
2. Dominant: which do not discriminate between homo and

heterozygotes
• They can be visualized by:
a. Gel electrophoresis
b. Ethidium bromide or silver staining
c. Radioactive or colorimetric probes

Comparison between co-dominant & dominant markers
Classification of molecular markers
• RAPD: Random Amplified Polymorphic DNA
• RFLP: Restriction Fragment Length Polymorphism
• AFLP: Amplified Fragment Length Polymorphism
• SSR: Simple Sequence Repeat
• ISSR: Inter Simple Sequence Repeat
• SNP: Single Nucleotide Polymorphisms
• SCAR: Sequence Characterized Amplified Region
• CAPS: Cleaved Amplified Polymorphic Sequence
• TRAP: Target Region Amplification Polymorphism
• DArT: Diversity Arrays Technology
• DAF: DNA Amplification Fingerprinting
• SRAP: Sequence Related Amplified Polymorphism

Types of DNA markers
On the basis of principles and methods of detection:
A. Hybridization based markers
RFLP: Restriction Fragment Length Polymorphism
• These markers are based on polymorphism arising due to
chromosomal aberrations occurred in the specific regions of DNA
• They are co-dominant; they can identify a unique locus as c-DNA
of known function used as probe, chromosomal positions of the
specific gene identified
• Restriction enzyme can recognize and cut DNA wherever a
specific short sequence occurs
• Molecular probe for Southern hybridization may be radioactive or
non radioactive
• Size of defined restriction fragments can be compared
electrophoretically
B.PCR based markers
• These markers reduce time, efforts and expenses
• They are based on use of pair of primers (reverse and forward)
• They are designed either on the basis of random sequences or on

specific sequences flanking the DNA segment that needs to be
amplified
1. Single primer used as forward and reverse primer – AP-

PCR(~20nt), RAPD(~10nt), DAF (~6-8nt) (same random primer is
inversed within an amplifiable distance)
2. A pair of primers used – STSs, SCARs or STARs

• Primer are short DNA sequences having free 3’-OH (~20bp)
usually used to amplify the marker loci
RAPD: Random Amplified Polymorphic DNA
• It was the 1st PCR based marker technique and it is by far the
simplest; 1st time reported by Welsh and McClelland (AP-PCR) &
Williams et al. in 1990
• Short PCR primers (approx. 10 bp) are randomly selected to

amplify random DNA segments throughout the genome
• The resulting amplification product is generated at the region

flanking a part of the 10 bp priming sites in the appropriate
orientation
• RAPD is a dominant marker; RAPD products are usually visualized

on Agarose gels stained with ethidium bromide
• Modified form of RAPD: DNA Amplification Fingerprinting (DAF)

Fig. Major events in RAPD
AFLP: Amplified Fragment Length Polymorphism
• They are differences in restriction fragment lengths caused by
• SNPs and INDELs that create/abolish restriction sites
• They are based on selective PCR amplification of restriction

fragments from a total digest of genomic DNA
• Oligonucletotide adapters (~20 bp) are ligated at the end of DNA

fragments
• Adapted DNA fragments are amplified by PCR

Fig. Major events in AFLP
SCAR: Sequence Characterized Amplified Region
• They are based on sequence of polymorphic

bands from RAPD/RFLP/AFLP linked to trait of
interest
• Longer primers (15-30 bp) are designed for

specific amplification of particular locus
• Higher in reproducibility than RAPD/RFLP
• These markers are co-dominant

SNP: Single Nucleotide Polymorphism
• SNP is a DNA sequence variation
occurring when a single nucleotide
(A, T, C, or G) in the genome differs
between members of a species
• E.g. AAGCCTA and AAGCTTA (called
alleles C and T)
• These markers can be known only
after DNA sequencing of several
genomes
• Used in biomedical research, crop
and livestock breeding programs
SSR: Simple Sequence Repeats
• These markers are also known as Short Tandem Repeat
(STRs)/microsatellites which are repeating sequences of 2-6 nt of
DNA (motifs)
• They are co-dominant, abundant, dispersed throughout the
genome
• They occur in DNA when a pattern of two or more nucleotides are
repeated which are adjacent to each other
• E.g. (CATG)n in a genomic region is typically in the non-coding
intron region
• Of four types: EST-SSR, Genomic-SSRs, Mitochondrial-SSRs,
Chloroplastic-SSRs
• Expressed Sequence Tags are the expressed regions in a DNA
sequence from a c-DNA clone that corresponds to m RNA; As they
represent the transcribed part of genome they show higher
transferability
• They are used for developing STSs, SSR, SNPs

ISSR: Inter-Simple Sequence Repeats
• The generation of ISSR markers involve PCR amplification of DNA

using a single primer composed of a microsatellite repeated
sequence
• In some cases primer also contains 1-4 base anchor at either 3′

or 5′ or at both ends, which target a subset of “simple sequence
repeats” (SSRs) and amplify the region between two closely
spaced and oppositely oriented SSRs
• ISSR technique permits the detection of polymorphisms in

microsatellites and inter-microsatellites loci without previous
knowledge of the DNA sequence
Applications of Molecular Markers
1. Confirmation of hybridity
2. Linkage Mapping
3. Marker Assisted Selection Role
of
4. Trait based Selection
Molecular Markers
5. Orthologous gene mapping in
Crop Improvement
6. Gene tagging Programs
7. Heterosis breeding
8. DNA fingerprinting for varietal identification
9. Phylogenetic and evolutionary studies
1. Confirmation of hybridity
• Heterozygosity of F1can be detected using molecular markers
2. Linkage Mapping
• For linkage mapping we want mapping population which is
immortal, universal, homozygous (true breeding type) and does
not fluctuate
• BC1F2, F2, DH, F2:F3, RILs, NILs
3. Marker Assisted Selection (MAS)
• MAS consists of identifying association between molecular
markers and genes controlling agronomic traits (major genes) ,
and using these to improve plant populations
• Selection is made on genotype rather than phenotype, which

increases the speed and efficiency of selection
• It is used for manipulating both qualitative (e.g. disease

resistance) and quantitative (yield) traits
• Molecular markers are used to increase the probability of

identifying truly superior genotypes, by early elimination of
inferior genotypes
4. Trait based selection
• Earliest demonstrations of power of molecular markers was
provided by Beckman and Soller (1986) for indirect selection of
gene in breeding program
• In South Australian Barley, a single dominant resistance gene to

cereal cyst nematode (Ha2) was transferred from a resistance to
susceptible variety through 3 cycles of marker assisted
backcrossing using a single molecular marker
5. Orthologous gene mapping
• Also known as “Comparitive mapping”
• cDNA clones of one crop plant are being mapped onto the
linkage maps of other crops
• More saturated maps can be generated by mapping marker clones
• 1st comparative maps in plants were generated by Tanksley in

Tomato & potato in 1988
• Generated especially in grasses

6. Gene Tagging
• Refers to mapping of genes of agricultural importance present
close to known markers
• Molecular marker very closely linked to a gene can act as a “tag”
• Gene tagging is the pre-requisite for MAS and Map based gene
cloning
• E.g. Cereal cyst nematode were tagged in wheat, Leaf rust

resistance genes Lr9, Lr10, Lr19, Fertility restorer genes for Polima
and Ogura cytoplasm in Brassica
7. Heterosis breeding
• Lee et al (1989) in corn suggested that RFLP analysis provides an
alternative to field testing
• Since then several attempts were made to correlate heterosis with

variability at molecular level
• Melchinger et al (1991) analyzed 32 maize inbred lines for

heterosis
• Stuber et al (1992) mapped QTLs contributing to heterosis in the

cross between elite maize inbred lines B73 and Mo17
• Xio et al (1995) mapped QTLs for heterosis in one of the highest

yielding indica x japonica hybrids and proposed domiance as the
major cause of heterosis in rice
8. DNA Fingerprinting for varietal identification
• Smith and Smith (1992) have presented comprehensive review on
need of fingerprinting of crop varieties
• RAPD, microsatellite and AFLPs are the markers of choice for

purpose because all are PCR based markers and does not require
any prior information on nucleotides
• It is useful in protection of proprietry germplasm especially the

CMS (Cytoplasmic Male Sterility) lines for hybrid seed production,
characterization of accessions in plant germplasm collections
• E.g. Sex identification in dioecious plants by Parasnis et al. (1999)

using microsatellite markers (GATA)4
9. Phylogenetic and Evolutionary Studies
• Molecular markers are used to study the evolutionary
relationships within and between species, genera or larger
taxonomic groupings
• Studies are made on similarities and differences among taxa using

numerous genetic markers especially RAPD and RFLP (Paterson et
al, 1991)
• E.g. Two lines of rice Azueena and PR 304 were classified as

indicas using morphological characters while these behaved as
japonicas in crossing studies

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Chapter 12 Molecular Markers

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Chapter 12: Molecular Markers

Their Application in Crop

• Molecular marker are based on naturally occurring changes or

• All molecular markers occupy specific genomic positions within

• Markers located in close proximity to desirable genes (tightly

• Agriculturally important traits are governed by many genes

Molecular markers are used because:

 they are selectively neutral as they are present in the non

 Makers are co-segregating with the trait of interest

 They follow exactly the Mendelian pattern of inheritance

 They are free from epistatic interaction or pleiotropic effect

 Closely linked to the trait of interest

 Enzyme based: Isozyme

• First true biochemical marker was an allelic variant of enzyme

• Isoforms can be resolved by gel electrophoresis based on their

• Can be easily assayed and detected

• They are low in polymorphism as compared to DNA markers

1. Co-dominant: discriminate between homo and heterozygotes

2. Dominant: which do not discriminate between homo and

• They can be visualized by:

b. Ethidium bromide or silver staining

c. Radioactive or colorimetric probes

• RFLP: Restriction Fragment Length Polymorphism

• AFLP: Amplified Fragment Length Polymorphism

• SSR: Simple Sequence Repeat

• ISSR: Inter Simple Sequence Repeat

• SNP: Single Nucleotide Polymorphisms

• SCAR: Sequence Characterized Amplified Region

• CAPS: Cleaved Amplified Polymorphic Sequence

• TRAP: Target Region Amplification Polymorphism

• DArT: Diversity Arrays Technology

• DAF: DNA Amplification Fingerprinting

• SRAP: Sequence Related Amplified Polymorphism

• They are based on use of pair of primers (reverse and forward)

• They are designed either on the basis of random sequences or on

1. Single primer used as forward and reverse primer – AP-

2. A pair of primers used – STSs, SCARs or STARs

• Short PCR primers (approx. 10 bp) are randomly selected to

• The resulting amplification product is generated at the region

• RAPD is a dominant marker; RAPD products are usually visualized

• Modified form of RAPD: DNA Amplification Fingerprinting (DAF)

• They are differences in restriction fragment lengths caused by

• SNPs and INDELs that create/abolish restriction sites

• They are based on selective PCR amplification of restriction

• Oligonucletotide adapters (~20 bp) are ligated at the end of DNA

• Adapted DNA fragments are amplified by PCR

• They are based on sequence of polymorphic

• Longer primers (15-30 bp) are designed for

• Higher in reproducibility than RAPD/RFLP

• These markers are co-dominant

• They are used for developing STSs, SSR, SNPs

• The generation of ISSR markers involve PCR amplification of DNA

• In some cases primer also contains 1-4 base anchor at either 3′

• ISSR technique permits the detection of polymorphisms in

• Selection is made on genotype rather than phenotype, which

• It is used for manipulating both qualitative (e.g. disease

• Molecular markers are used to increase the probability of

• In South Australian Barley, a single dominant resistance gene to

• More saturated maps can be generated by mapping marker clones

• 1st comparative maps in plants were generated by Tanksley in