Chapter 4: Molecular markers and their application in

plant improvement
4.1.Common terminologies related to molecular markers
4.2. Introduction to molecular markers
4.3.Types of markers
4.4. DNA fingerprinting in plant improvement
4.5. trait linked DNA based molecular markers in plant
4.1.Common terminologies related to molecular markers
Centimorgan/cM: genetic distance b/n a chromosome between two loci.
Codominant Marker: distinguish the homozygote and heterozygote
Codominant In heterozygotes: when contributions of both alleles seen
in phenotype.
DNA Hybridization: A process that combines two complementary
single-stranded DNA molecules to form double-stranded molecule
through base pairing.
Dominant Marker: A marker that cannot distinguish the homozygous
and heterozygous states.
Dominant: A genetic phenomenon in which the phenotypes of dominant
homozygotes (AA) and heterozygotes (Aa) are the same
Genetic or Linkage Map: A map that shows the relative locations of
genetic or molecular markers calculated using cross-over rates..
Isozyme: protein markers have the same activities but can be
distinguished by different electrophoresis speeds.
DNA Library: A gene set /cDNA /genome that contains clones of DNA
from a target crop.
Linkage: genetic proximity of two or more genes on a chromosome.
Locus: The location of a gene on a chromosome or chromosome map.
Physical Map: A map that showing the positions of sequence features
using DNA sequence data.
Polymorphism: phenomenon in which more than one different allele
exists at the same locus & observed at phenotype, protein/ DNA level.
Probe: A DNA fragment that complementarily bonds to a specific DNA
sequence.
 Genetic Marker
 Genetic Marker: A marker that is closely linked with useful traits,
such as disease resistance or abiotic stress resistance.

 When a specific phenotype abiotic or biotic stress condition is

difficult to determine, Genetic markers are a viable alternative.

 Genetic markers can be classified into:

 morphological markers

Molecular marker,
 protein markers,
 DNA markers
 Morphological markers
 These markers depend on phenotypic data like color, size shape etc

 They are easy to assay with low cost

 However they have more limitations:

 their potential numbers are low b/se
 They are influenced by environmental factor and growth stage

 Morphological markers were used first for genetic analysis by

geneticists like Gregor Mendel and Thomas Hunt Morgan.
 Molecular markers
 It indicates technology that uses phenotype determining or closely
related genes to find similarities/ differences among individual
plants

 By analyzing molecular markers, individual plants with useful

phenotypes can be selected at the seedling stage.

 Molecular markers for plant breeding are based on genetic

differences among individuals in alleles at a certain locus.

 Molecular marker can be:

protein markers or
DNA markers
protein markers
 protein markers could be analyzed with low cost.
 Different alleles encode proteins different in sizes or biochemical
characteristics that can be observed & used as markers.
 Isozymes are an example,
 They having same enzymatic activity.
 Isozymes are useful protein markers because
can be distinguished from each other based on differences in
charge/size,
 can be easily detected by separating via gel electrophoresis
isozyme produces color from substrate, producing band on
the gel.
 isozyme analysis is very cheap and simple
 used for studies of maize, wheat, and barley decades before
DNA markers were developed.
Drawback of protein markers
 limited number of possible markers (only a few dozen developed),
 they are not distributed evenly on the chromosome,
 often the enzyme activity depends on the plant’s age or tissue type.
 DNA Markers
 They use sequence differences among species or individuals within
a species.

 Genetic differences among individuals in a group are usually due

recombination that rearranges the chromosomes,

 for example, insertions, deletions, inversions, translocation

events, or reduplication.

 Chromosomal rearrangements can vary in size, from just a few

base pairs to millions.

 DNA mutations in the form of base pair substitutions also occur.

 To develop genetic markers using DNA variants, DNA hybridization

or PCR techniques are often used.
 Types of DNA markers
 DNA markers can be hybridization or PCR techniques based

DNA Markers Using Hybridization

 In DNA hybridization, a short DNA fragment that is homologous to

the target DNA is used as a probe.

 The probe is tagged with a radioisotope and hybridized with the

DNA being analyzed.

 DNA variations can be detected based on the target–probe

hybridization or the size of the hybridized DNA fragment.
 RFLP is an example.

 RFLP : classic example of molecular markers using DNA

hybridization,

 RLFPs are a first-generation technique and the basis of

Restriction enzymes and Southern blotting
Restriction Endonucleases:
 they recognize a specific restriction DNA sequence & cut the DNA

 The recognized sequence can be four, six, or eight base pairs in

length, depending on the enzyme.

 individual genotypes, even within a species, result in different

patterns of fragmentation.

 However, when too many fragments are generated, they cannot be

differentiated by typical electrophoresis.

 Even when they can be visualized, identifying homologous

fragments in different individuals is nearly impossible.
So, Southern blotting analysis was developed to overcome
these problems.
 Southern Blot Analysis
 Southern blot analysis uses 0.5–3.0 kb DNA fragments as probes to
detect other fragments
 The probe is selected from a genomic DNA library, a cDNA
library, or DNA fragments related to the gene(s) of interest.
 Southern blot analysis proceeds as :
1. DNA is extracted from the plant materials
2. DNA fragments are digested with restriction enzymes and
separated by size using agarose gel electrophoresis.
3. the gel is treated in a strong alkali solution to denature the DNA
strands.
4. fragments are transferred to nitrocellulose/nylon membrane &
exposed to UV light/heat to fix them onto membrane.
5. The DNA probe is labeled with radioactive isotope or fluorescent dye
and hybridized to the membrane-fixed DNA fragments.
6. the membrane is washed to remove unbonded bonded probes. The washed
membrane is exposed to X-ray film to visualize hybridization band
Molecular Markers Using PCR
 PCR techniques require only a small amount of DNA and are
relatively simple and inexpensive.
 They include using :
 minisatellites
 microsatellites,
 sequence-specific primers
such as sequence tagged sites (STSs)
expressed sequence tags (ESTs),
 random primers
 such as random amplified polymorphic DNA (RAPD) or
amplified fragment length polymorphism (AFLP) to amplify
the DNA fragment and analyze its variants.
Minisatellites
 are 9–100 bp DNA sequences that are repeated along the
genome.

The number of repeats varies but is usually under 1000.

Polymorphism in the number of repeats is called variable

number of tandem repeats (VNTR).

PCR using primers that flank the repeat allow size variation of
amplicon, and thus VNTR, to be detected.
Microsatellites
 They are called simple sequence repeats (SSRs),
 1–6-bp random sequences that are repeated fewer than 100 times.
 The most common case is two alternating bases, such as
(CA)n:(GT)n, (CG)n:(GC)n, or (AT)n:(TA)n.
 Three or four base-pair repeats also exist, but are less common.
 When a microsatellite repeat is discovered, the flanking sequences
can be used as PCR primers to develop SSR markers.
 In the past, to develop an SSR marker, a probe containing a repeat
sequence was first used to identify homolog repeats in genes from a
DNA library.
 However, because large amounts of sequence data are now easy to
obtain, the need to screen a DNA library no longer exists.
 SSR markers can now be developed from gene bank data.
 After a repeat is identified, flanking sequence is used to amplify SSR,
and length differences among amplicons reveal SSR polymorphisms.
 SSR markers are codominant, easy to analyze, and provide
reproducible results, making them ideal molecular markers.
Random Amplifi ed Polymorphic DNA (RAPD) Markers
 RAPD markers can be developed even without DNA sequence data
of the target species.
 These markers are generated using random PCR primers of about
10 bp in length.
 random primers can bind with any complementary sites in the
genome
 the PCR product can be analyzed by electrophoresis.
 DNA sequence differences among individuals at priming sites
cause different band patterns, allowing polymorphisms on agarose gel
 RAPD markers are relatively easy to develop, and their results are
relatively simple to analyze.
 However, because RAPD markers are inherited dominantly and
experimental reproducibility can vary with reaction conditions,
precise genotype analysis is difficult.
Amplified Fragment Length Polymorphism (AFLP) Markers
 To solve the reproducibility problems of RADP, the Dutch biotech
company KeyGene developed the AFLP method in 1995.
 In AFLP, restriction-digested DNA fragments are amplified using
PCR.
 The basic principle of AFLP :
 First, DNA is digested by two different restriction enzymes.
 An adaptor, a small double stranded oligonucleotide that
complements the sticky end of the digest,
 Then, the adaptor-attached DNA fragments are amplified by
PCR using primers that complement the adaptors.
 One primer must be labeled with a radioactive isotope to
visualize the amplified products.
 A few additional base pairs (selective nucleotides) can be added to
the primer sequence beyond the adaptor region to decrease the
number of amplified fragments.
 For e.g, if 3 selective nucleotides are added to each primer,
number of amplified fragments will be reduced by 1/46.
Single Nucleotide Polymorphism (SNP) Markers
 SNPs are literally single-base differences among individuals and
represent 80 % of DNA polymorphisms.
 On average, one SNP exists every 1 kb in the human genome

 The most direct way of identifying SNPs in genomes is by directly

comparing DNA sequences.

 Today, thanks to the accumulation of DNA sequence data, SNP

markers can be developed in silico by analyzing existing databases.
 Once SNPs are identified, differences among individuals can be
analyzed as:
 Allele-specific PCR: When one of the two primers used in PCR
has a 3′ end that does not complement the template DNA, DNA
amplification is slower than usual.
 Therefore, if the SNP is set as the 3′-end of the primer sequence,
one allele will amplify well, while different alleles will not,
allowing easy allele distinction.
DNA Chip
 One way to test SNPs. Artificial 25 bp oligonucleotides are attached
to glass or metal plates.
 Fluorescently-labeled probes of DNA fragments are hybridized to
determine which alleles have a given SNP
Pros and Cons of Various DNA Marker Methods
 The ideal DNA marker would have the following characteristics:
 even distribution throughout the genome,
 high experimental reproducibility,
 fast and cheap analysis,
 little DNA requirement, and
 linkage with various phenotypes.

 Currently, no DNA markers meet all of these conditions; each

technique has pros and cons that must be considered when
planning experiments so the selected markers match the
purpose of the experiment.
 Comparison of DNA markers
Chapter 4: Molecular markers and their application in
plant improvement
4.1.Common terminologies related to molecular markers
4.2. Introduction to molecular markers
4.3.Types of markers
4.4. DNA fingerprinting in plant improvement
4.5. trait linked DNA based molecular markers in plant
4.4. DNA fingerprinting for plant improvement
What is DNA Fingerprinting
 Many disciplines in botany are dependent on ability to differentiate
among plant genotypes for d/t purpose.

 Traditionally it conducted mainly through data on morphological

characteristics but these have certain limitations

 A DNA fingerprint is a genetic photograph of an individual,

whether that individual is a plant, animal or person.

 technique of DNA finger printing has been developed by Alec

Jeffreys using the science of genetics for forensic purpose.
 What Type of Genetic Variation observed
during DNA fingerprint?
•Length Variation
short tandem repeats (STRs)
CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT

•Sequence Variation
single nucleotide polymorphisms (SNPs)
insertions/deletions
GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC
Steps in DNA fingerprint Analysis

Sample Collection

DNA Extraction

Genotyping By
selected marker

Interpretation of
Results

Store in biological
Database
Hybridization-based restriction fragment length polymorphism
fingerprints of tomato plants (Solanum lycopersicum).
.
Random amplified polymorphic DNA (RAPD) fingerprints of
Pelargonium.
Relevance of Fingerprinting in plant improvement
 DNA finger printing for diversity study
 assessing genetic diversity improved by the use of DNA
fingerprinting for estimating population genetics parameters like
expected heterozygosity to start breeding
 DNA finger printing for new variety release
Novelty, distinctness, uniformity and stability are essential
requirements of the grant of protection to all the varieties.
The DNA fingerprinting is a robust tool in identification of
crop varieties frequently developed by the plant breeders.
 4.5.trait linked DNA based molecular markers in plant
Developing Trait-Linked Molecular Markers

 To check whether a molecular marker and target trait are linked,

marker can be developed by generating a linkage map.

 number of recombinant individuals is counted & genetic distance

b/n marker & target gene can be calculated for linkage map.

 So, developing of trait- linked marker can be considered

When Linkage Maps/ Biological Data Not Available

When Genetic Linkage Maps / Biological Data Available

When Genetic Linkage Maps /Biological Data Are Not
Available

 When biological data were insufficient several maping population

my be used to develop trait linked markers, such as:
 Near Isogenic Lines (NILs)
 bulked segregant analysis (BSA)

Then Molecular markers such as RAPD or AFLP can be used.

Near Isogenic Lines
 the genomes of NILs differ only in the region near the target trait.
 NILs can be developed using backcrossing .
1. A ‘donor’ (P1) with the target gene is crossed with the ‘recurrent’
parent (P2) to obtain the F1 generation.
2. The F1 and P2 are backcrossed to make the next generation.
3. individuals with target trait are chosen to be backcrossed again.
4. A line that is homozygous for the target gene is selected to
develop an NIL through 1–2 rounds of self-fertilization.
5. To obtain NIL, plants should be advanced to the BC4F2–BC5F3
generation.
Near isogenic line (NIL) development scheme
Bulked Segregant Analysis (BSA)
 BSA is a method can be used when NILs is not available.
 genomic DNA of several plants with the same phenotype is bulked
into one pool.
 Two bulked pools of segregants differing for one trait will differ only
at the locus harboring that trait.
 BSA can be performed via the following steps:
1. Cross target gene-containing P1 & gene-lacking P2 to generate F1
generation & self-pollinate F1 to get the F2 generation.
2. Determine phenotype for target gene l& extract DNA.
3. Dilute DNA to the same concentration,
4. then mix the DNA samples of the same phenotype to make a DNA
‘bulk’ for each phenotype.
Analysis of BSA for marker development
 If one assumes that both parents have a homozygous genotype at
all loci, the genotypes of all F1 individuals will be the same.
 Consider two loci, B and C, that are far from the target gene.
 After performing BSA and making DNA bulks, there must be an
AA bulk and an aa bulk.
 While the allele frequency for gene A in the AA bulk is A:a = 1:0,
 for other loci that are not linked to A, the genotype frequency will
be BB:Bb:bb = CC:Cc:cc = 1:2:1.
 Thus, the allele frequency for other loci is 1:1, showing
heterozygotes as a result
BSA scheme
When Biological Data Are Available

 Many genes have been identified with their functions in various

organisms.

 This biological information can be used to discover the functions of

genes in other species.

 NGS technologies allowing entire genomes sequence as

references & resequencing individuals of same species common.

 Such abundant biological information allows new molecular markers

to be developed with speed and accuracy

 Methods to develop molecular markers depend on whether existing

biological information

 When there is abundant biological information about a crop

markers develop by candidate gene/ comparative genetics .
Using Comparative Genetics
 Comparative genetics is based on evolution as that any group of
organisms have a common ancestor
 Species that recently shared a common ancestor have similar
numbers of chromosomes, and gene loci are also similar/ synteny.
 The more distantly related two species are, the less commonality
in their DNA,
 so comparative genetics is usually applied to species in the same
genus or, in some cases to species within a family.

• Phylogenic tree of six species or

originated from a common ancestor
 Chromosome map used in comparative genetics showing
synteny between two phylogenetically close species
Sequence data
 If complete genome or EST sequence data exist for the species used
for comparison via d/t research ways

 If the entire genome sequence is known for a model species & only
EST sequence data exist for the target plant,

the ESTs can be used as common markers because they

originate from expressed genes.
EST sequences are highly likely to be conserved between
the two species,
similar EST sequences can be assumed to be present in the
genome of a model plant.
BLAST
 Basic Logical Alignment Search Tool (BLAST) is one fundamental
program for analyzing nucleotide sequences.
 BLAST compares a submitted sequence with in database
 BLAST has many embedded algorithms, including blastn, blastp,
blastx, tblastn, and tblastx.
Finding Functional Candidate Genes
 Candidate genes can be divided into functional and positional
candidates.

 In plants, the sequence, structure, and function of various genes have

been discovered through research using model plants such as
Arabidopsis, rice, and tomato.

 a gene with a known function in one species will likely have a

similar function in another.

 This inference justifies the development of molecular markers using

functional candidate genes.

 Candidate genes found in EST or whole-genome DBs by BLAST

should be confirmed experimentally.
General Strategies for Developing Molecular Markers
 Modern comparative genetics does not simply compare genetic
maps/ sequences but all information used to develop markers.

 Inexpensive systems that analyze gene expression by sequencing

transcriptomes with NGS technology are already available.

 Enormous genetic data, with functions identified, can be found at

NCBI, and cab be analyze by the application of bioinformatics

 To develop target-gene-related molecular markers, the phenotype

information and molecular marker genotype should be known

 In the big-data era, analyzing a large amount of data with quick

homemade scripts will be needed for developing molecular
markers.
 Marker Assisted selection (MAS)
 It refers to use of DNA markers that are tightly-linked to target
loci as a substitute for or to assist phenotypic screening

 Assumption: DNA markers can reliably predict phenotype

Advantages of MAS
 Simpler method compared to phenotypic screening
 Especially for traits with laborious screening
 Save time and accelerated variety development
 Selection at seedling stage
 Important for traits such as grain quality
 Can select before transplanting in some crop like rice
 Increased reliability
 No environmental effects
 Can discriminate between homozygotes and heterozygotes and
select single plants
 MARKER-ASSISTED BREEDING

P1 x P2
Susceptible Resistant

F1

large populations consisting of thousands

F2
of plants

MARKER-ASSISTED SELECTION (MAS)

Method whereby phenotypic selection is based on DNA markers