Molecular markers

molecular markers and their advantages

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INTRODUCTION
Those characters which can be easily identified are called marker characters.
Any genetic element (locus, allele, DNA sequence or chromosome feature) which can be readily detected by
phenotype, cytological or molecular techniques, and used to follow a chromosome or chromosomal segment
during genetic analysis is referred to as marker.
GENETIC MARKERS
Any traits of an organism that can be identified with confidence and relativeease,andcanbefollowedina
mapping population is called genetic marker.
It can be detected with naked eye, as differences in electrophoretic mobility of specific proteins, or as differences 
in specific DNA sequences.
Markers are of four types, viz:
(i) Morphological,
(ii) Biochemical,
(iii) Cytological, and
(iv) DNA markers.
1.Morphological marker
also called naked eye polymorphism.
In plant breeding, markers that are related to variation in shape, size, colour and surface of various plant parts are
called morphological markers. Such markers refer to available gene loci that have obvious impact on morphology
of plant. Genes that affect form, coloration, male sterility or resistance among others have been analyzed in many
plant species.
In rice, examples of this type of marker may include the presence or absence of awn, leaf sheath coloration, height,
grain color, aroma etc. In well-characterized crops like maize, tomato, pea, barley or wheat, tens or even hundreds
of such genes have been assigned to different chromosomes.
Advantages
a) NEP traits represents the actual phenotypes of importance to us, while protein and DNA markers are important only as 
arbitrary loci used for linkage mapping and often do not Correspond directly to specific phenotypes.
b) NEPs factors can be scored quickly, simply, without laboratory equipment.
Disadvantages
a. They generally express late into the development of an organism. Hence their detection is dependent on the development 
stage of the organism.
b. They usually exhibit dominance.
c. Sometimes they exhibit deleterious effects.
d. They exhibit pleiotropy.
e. They exhibit epistasis.
f. They exhibit less polymorphism.
g. They are highly influenced by the environmental factors.

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ii. Cytological markers
Markers that are related to variation in chromosome number, shape, size and banding pattern are referred to as
cytological markers. In other words, it refers to the chromosomal banding produced by different stains;
for example, G bandingor Giemsa banding is a technique used in cytogenetics to produce a visible karyotype by staining 
condensed chromosomes. It is useful for identifying genetic diseases through the photographic representation of the 
entire chromosome complement..
advantages of cytological marker
1. Readily available.
2. Requires small equipments.
Disadvantages of cytological marker
1. Limited in number
2. They exhibit less polymorphism, need experts.
iii. Biochemical markers 
Monoterpenes
Monoterpenes are a subgroup of the terpenoid substances found in resins and essential
oils of plants ,Although the metabolic functions of monoterpenes are not fully understood, they probably play an important 
role in resistance to attack by diseases and insects (Hanover, 1992). The concentrations of different monoterpenes, such as 
alpha‐pinene, beta‐pinene, myrcene, 3‐carene and limonene are determined by gas chromotography and are useful as 
genetic markers.
Advantage
1. Monoterpene genetic markers have been applied primarily to taxonomic and evolutionary
studies.
2.   Although, monoterpenes were the best available genetic markers for forest trees in the 1960s and
early 1970s. 
Demerits
they require specialized and expensive equipment for assay.
there are relatively few monoterpene marker loci available and most express some form of
dominance in their phenotypes.

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Allozymes
Monoterpenes were gradually replaced by allozyme genetic markers because. Allozymes have been the most important type 
of genetic marker in forestry and are used in many species for many different applications.
Allozymes are allelic forms of enzymes that can be distinguished by a procedure called electrophoresis. The more general term 
for allozymes is isozymes, and refers to any variant form of an enzyme, whereas allozyme implies a genetic basis for the variant
form.
Most allozyme genetic markers have been derived from enzymes of intermediary metabolism, such as enzymes in the 
glycolytic pathway.
Advantages
Allozyme analysis is fairly easy to apply and standard protocols for its use in trees are available.
allozymes are less expensive to apply, are codominant in expression, and many more marker loci can be assayed.
Isozyme markers
Another type of protein‐based genetic marker utilizes two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Unlike 
allozymes where single known enzymes are assayed individually, the 2‐D PAGE technique simultaneously reveals all enzymes 
and other proteins present in the sample preparation.
The proteins are revealed as spots on gels and marker polymorphisms are detected as presence or absence of spots.
This technique has been used most extensively for linkage mapping in Pinus pinaster where protein polymorphisms have been
assayed from both seed and needle tissues.
advantage
many marker loci can be assayed simultaneously on a single gel.
Disadvantage
assays are more difficult than in allozyme analyses, and the markers are often dominant in their expression.
Advantage
1. Require simple equipments.
2. A vigorous complement to the morphological assessment of variation.
Disadvantages1. The main weakness of allozymes is their relatively low abundance.
2. Low level of polymorphism.
3. Allozymes are in fact phenotypic markers and they may be affected by 
environmental conditions.
4. Profiling of a particular allozyme markers may  change on the type of tissue 
used for the analysis.
However, the availability of useful protein markers is a limitation. 

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iv. Molecular marker
DNA is the genetic material. Each chromosome has about 10
8
‐10
10
base pairs but only 10% of the genome is actively engaged in
translation. The rest of the genetic material remains unnoticed in terms of character expression. The assessment of variation at
the DNA level provides a chance to map the genome. Specific regions on the DNA molecule both in the coding as well as non‐
coding regions can be identified as markers.
DNA markers are such polymorphic sequences seen in different individuals.
A considerable part of the DNA shows highly repetitive sequences in the non‐coding regions of DNA. The comparison of these 
regions can also provide valuable clues of genetic and evolutionary relationships. The DNA sequences can be cut with the help of
restriction endonucleases, analyzed with the help of DNA probes and electrophoretic procedures.
Definition
Any unique DNA sequence which can be used in DNA hybridization, PCR or restriction mapping experiments to identify that 
sequence is called DNA marker.
Properties of DNA Marker:
An ideal DNA marker should have some properties or characteristics.
Important properties of an ideal DNA markers
i. Polymorphism:
Markers should exhibit high level of polymorphism. In other words, there should be variability in the markers. It
should demonstrate measurable differences in expression between trait types and/or gene of interest.
ii. Co-Dominant:
Marker should be co-dominant. It means, there should be absence of intra-locus interaction. It helps in identification
of heterozygotes from homozygotes.
Advantages
They are not influenced by the environment.
They are more reliable (marker should be tightly linked to target loci, preferably less than 5cM genetic distance).
Level of polymorphism is high.
Easily excessable marker sequences.
Highly reproducible.
Multiple alleles for each markers.
They abundantly occur throughout the genome
They have simple inheritance (often co‐dominant).
They are easy and fast to detect.
They exhibit minimum pleiotropic effect.

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iii. Multi-Allelic:
The marker should be multi-allelic. It useful in getting more variability/ polymorphism for a character.
iv. No Epistasis:
There should be absence of epistasis. It makes Identification of all phenotypes (homo- and heterozygotes)
easy.
v. Neutral:
The marker should be neutral. The substitution of alleles at the marker locus should not alter the
phenotype of an individual. This property is found in almost all the DNA markers.
vi. No Effect of Environment:
Markers should be insensitive to environment. This property is also found in almost all the DNA markers.
Type of molecular markers
RFLP  (orRestriction fragment length polymorphism)
AFLP  (orAmplified fragment length polymorphism)
RAPD (orRandom amplification of polymorphic DNA)
SSRMicrosatellitepolymorphism, (orSimple sequence repeat)
SNP  (orSingle nucleotide polymorphism)
STS  (or sequence taged sites)
CAPS      (Cleaved amplified polymorphic sequence)
SCAR (Sequence Characterized Amplified Region )
1.Restriction fragment length polymorphism (RFLP):
RFLP was the very first technology employed for the detection of polymorphism, based on the DNA sequence
differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and re-combinations
of genomic DNA. An outline of the RFLP analysis is given in Fig.1, and schematically depicted in Fig.2.
The procedure basically involves the isolation of genomic DNA, its digestion by restriction enzymes, separation by
electrophoresis, and finally hybridization by incubating with cloned and labeled probes.

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Advantages of RFLPs 
•Highly robust methodology with good transferability between laboratories 
•Codominantly inherited and, as such, can estimate heterozygosity 
•No sequence information required,  Because based on sequence homology. 
•Highly recommended for phylogenetic analysis between related species 
•Well suited for constructing genetic linkage maps 
•Locus‐specific markers, which allow synteny studies( physical co‐localization 
of genetic loci on the same chromosome within an individual or species) 
•Discriminatory power—can be at the species and/or population levels (single‐
locus probes), or individual level (multi‐locus probes) 
•Simplicity—given the availability of suitable probes, the technique can readily be 
applied to any plant
•Disadvantages of RFLPs 
•Large amounts of DNA required 
•Automation not possible 
•Low levels of polymorphism in some species 
•Few loci detected per assay
•Need a suitable probe library
•Time consuming, especially with single‐copy probes 
•Costly 
•Distribution of probes to collaborating laboratories required
•Moderately demanding technically
•Different probe/enzyme combinations may be needed
Applications
•Genetic diversity
•Genetic relationships
•History of domestication
•Origin and evolution of species
•Genetic drift and selection
•Whole genome and comparative mapping
•Gene tagging
•Unlocking valuable genes from wild species
•Construction of exotic libraries
2.Random amplified polymorp hic DNA (RAPD) markers:
RAPD is a molecular marker based on PCR amplification. An outline of RAPD is depicted in fig.3. The DNA isolated
from the genome is denatured the template molecules are annealed with primers, and amplified by PCR.
Single short oligonucleotide primers (usually a 10-base primer) can be arbitrarily selected and used for the
amplification DNA segments of the genome (which may be in distributed throughout the genome). The amplified
products are separated on electrophoresis and identified.

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Advantages
•It can be used with uncharacterized genomes
•can be applied to cases in which only small quantities of DNA are available
•Absolutely no knowledge of the target genome is required
•It can be used on any DNA sample.
•RAPD is an inexpensive yet powerful tool for typing of bacterial species.
•Even if the same primer is used with different samples, they will produce different results and different bands
patterns that may allow for recognition of the various strains.
•Can be used to study genetic polymorphism between closely related species.
•Can be used to select variants of microbial isolates.
Limitations
•The primer sequence has to be right to produce the right results.
•Since the primer targeting is random, it is absolutely essential to have a large genome template.
•Minute quantities of available DNA or degraded DNA samples cannot be subjected to RAPD (unlike PCR).
•It is very likely that the primer will not be able to find complementary sequences on the target.
•It has a low power of resolution unlike other methods of DNA analysis.
•The quality of the DNA used in the reaction will affect the outcome.
•The concentration of PCR reagents, their purity, and the conditions in which the reaction is carried out will all
affect the results.
•RAPD requires knowledgeable and careful operation for good results and for these results to be reproducible.
•If there is a mismatch between the primer and the template DNA, there will be reduced product or even no PCR
product. Hence, reading the results can be a little tricky sometimes.
•If the 3' ends of the primer are not facing each other there will be little or no product.
•Mutation of target DNA can give wrong profile.
3.Amplified fragment length polymorphism
(AFLP):
AFLP is a novel technique involving a combination of RFLP and RAPD. AFLP
is based on the principle of generation of DNA fragments using restriction
enzymes and oligonucleotide adaptors (or linkers), and their amplification by
PCR. Thus, this technique combines the usefulness of restriction digestion
and PCR.
The DNA of the genome is extracted. It is subjected to restriction digestion by two 
enzymes (a rare cutter e.g. Msel; a frequent cutter e.g. EcoRI). The cut ends on both 
sides are then ligated to known sequences of oligonucleotides.
PCR is now performed for the pre‐selection of a fragment of DNA which has a single 
specific nucleotide. By this approach of pre‐selective amplification, the pool of 
fragments can be reduced from the original mixture. In the second round of 
amplification by PCR, three nucleotide sequences are amplified.
This further reduces the pool of DNA fragments to a manageable level (< 100). 
Autoradiography can be performed for the detection of DNA fragments. Use of 
radiolabeled primers and fluorescently labeled fragments quickens AFLP.

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Advantages of AFLPs 
•AFLPs allow a quick scan of the whole genome for polymorphisms
•Because of the large number of bands generated, each marker gives a highly informative fingerprint
•They are also highly reproducible
•No prior sequence information or probe generation is needed
•Extremely useful in creating quick genetic maps. 
Disadvantages of AFLPs 
•AFLPs generate huge quantities of information, which may need automated analysis and therefore computer technology 
•AFLP markers display dominance
•In genetic mapping, AFLPs often cluster at the centromeres and telomeres
•They are technically demanding in the laboratory and, especially, in data analysis
Applications
•Genetic diversity assessment 
•Genetic distance analysis 
•Genetic fingerprinting 
•Analysis of germplasm collections  
•Genome mapping 
•Monitoring diagnostic markers 
4. Sequence‐Tagged Site (STS)
is a relatively short, easily PCR‐amplified sequence (200 to 500 bp) which can be specifically amplified byPCRand detected in 
the presence of all other genomic sequences and whose location in the genome is mapped.
The STS concept was introduced by Olsonet al. (1989).
In assessing the likely impact of the Polymerase Chain Reaction (PCR) on human genome research, they recognized that single‐
copy DNA sequences of known map location could serve as markers for genetic and physical mapping of genes along the 
chromosome.
The advantage of STSs over other mapping landmarks is that the means of testing for the presence of a particular STS can be 
completely described as information in a database: anyone who wishes to make copies of the marker would simply look up the 
STS in the database, synthesize the specified primers, and run the PCR under specified conditions to amplify the STS from 
genomic DNA.
STS‐based PCR produces a simple and reproducible pattern on agarose or polyacrylamide gel. In most cases STS markers are co‐
dominant, i.e., allow heterorozygotes to be distinguished from the two homozygotes.
The DNA sequence of an STS may contain repetitive elements, sequences that appear elsewhere in the genome, but as long as 
the sequences at both ends of the site are unique and conserved, researches can uniquely identify this portion of genome 
using tools usually present in any laboratory.
in broad sense, STS include such markers as microsatellites , SCARs, CAPs, and ISSRs.

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Microsatellites
Polymorphic loci present in nuclear DNA and organellar DNA that consist of repeating units of 1‐10 base pairs, 
most typically, 2‐3 bp in length, also called Simple Sequence Repeats (SSR), Sequence‐Tagged Microsatellite 
Sites (STMS) or Simple Sequence Repeats Polymorphisms (SSRP). SSRs are highly variable and evenly 
distributed throughout the genome. This type of repeated DNA is common in eukaryotes. These 
polymorphisms are identified by constructing PCR primers for the DNA flanking the microsatellite region. The 
flanking regions tend to be conserved within the species, although sometimes they may also be conserved in 
higher taxonomic levels.
Sequence Characterized Amplified Region (SCAR)
DNA fragments amplified by the Polymerase Chain Reaction (PCR) using specific 15-30 bpprimers, designed from
nucleotide sequences established in clonedRAPD(Random Amplified Polymorphic DNA) fragments linked to a trait of
interest. By using longer PCR primers, SCARs do not face the problem of low reproducibility generally encountered
with RAPDs. Obtaining a co-dominant marker may be an additional advantage of converting RAPDs into SCARs.

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Cleaved Amplified Polymorphic Sequences (CAPS)
polymorphisms are differences in restriction fragment lengths caused bySNPsorINDELsthat create or abolishrestriction 
endonucleaserecognition sites inPCRamplicons produced by locus‐specific oligonucleotideprimers.
The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to displayRFLP.
Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two 
different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain 
two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be 
obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel 
electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as 
doublets.
Advantages of CAPS
•Most CAPS markers are co‐dominant and locus‐specific.
•Most CAPS genotypes are easily scored and interpreted.
•CAPS markers are easily shared between laboratories.
•CAPS assay does not require the use of radioactive 
isotopes, and it is more amenable, therefore, to 
analyses in clinical settings.
single-nucleotide polymorphism(SNP)
It is a DNA sequence variation occurring when a single nucleotide adenine (A), thymine (T), cytosine (C), or guanine
(G]) in thegenome(or other shared sequence) differs between members of a species or pairedchromosomesin an
individual.
For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in
a single nucleotide. In this case we say that there are twoallelesC and T.
Almost all common SNPs have only two alleles.
advantages
•PCR products can be very small: – Markers will work with extremely degraded DNA samples
•More common in genome
•May possibly multiplex hundreds or thousands on one chip
•Sample processing may be completely automated 
•No stutter products
•disadvantages
•• Less alleles – Each marker is less informative
•• Therefore have to genotype many more SNPs to get same level of information about DNA sample
•• Mixture interpretation is more difficult
•• Multiplexes don’t actually work yet – Currently uses more of the DNA sample than STRs use

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Difference among DNA markers
MOLECULAR GENETIC MAPS AND THEIR 
APPLICATIONS IN PLANT BREEDING
•Molecular markers are useful in constructing high density genetic maps of crop.
•location of genes in relation to the markers used. Parallelism in gene order (gene synteny) and evolutionary 
relationships can be analyzed.
•Marker‐assisted early generation selection of transgressive segregants can increase the speed of developing 
new varieties. 
•Selective transfer of desirable genes from wild germplasm can be made easy, thus making introgression of 
genes easy.
•Mapping of quantitative trait loci (QTL) becomes easy with the help of molecular markers.
•Gene pyramiding (assembly of the polygenes responsible for a character) becomes easy with the help of 
molecular markers.
•DNA fingerprinting and characterization of crop varieties  and germplasm can be used as a very effective tool 
to analyze the genetic variability of the crop genetic resources available.  

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Mapping and tagging of genes: Generating tools for marker-assisted selection
in plant breeding
Plant improvement, either by natural selection or through the efforts of breeders, has always relied upon creating,
evaluating and selecting the right combination of alleles. The manipulation of a large number of genes is often
required for improvement of even the simplest of characteristics With the use of molecular markers it is now a
routine to trace valuable alleles in a segregating population and mapping them. These markers once mapped
enable dissection of the complex traits into component genetic units more precisely, thus providing breeders with
new tools to manage these complex units more efficiently in a breeding programme.
•first genome maps in plants was reported in maize , followed by rice,Arabidopsisetc. using RFLPmarkers. 
have since then been constructed for several other crops like potato, barley, banana, members of 
Brassicaceae, etc.
•RFLP markers have proved their importance as markers for gene tagging and are very useful in locating and 
manipulating quantitative trait loci (QTL) in a number of crops.
•The very first reports on gene tagging were from tomato, availing the means for identification of markers 
linked to genes involved in several traits like water use efficiency, resistance toFusarium oxysporum(the 12 
gene), leaf rust resistance genes LR 9 and 24, and root knot nematodes (Meliodogynesp.). 
Phylogeny and evolution
Most of the early theories of evolution were based on morphological and geographical variations 
between organisms. However, it is becoming more and more evident that the techniques from 
molecular biology hold a promise of providing detailed information about the genetic structure of 
natural population, than what we have been able to achieve in the past. RFLP, DNA sequencing, and 
a number of PCR‐based markers are being used extensively for reconstructing phylogenies of various 
species. 
RFLPs have been used in evolutionary studies for deducing the relationship between the hexaploid
genome of bread wheat and its ancestors.
Diversity analysis of exotic germplasm
genetic variation in crop plants has continued to narrow due to continuousselection pressure for specific traits i.e.
yield, . This risk was brought sharply into focus in 1970 with the outbreak ofsoutherncornleafblightwhich
drastically reduced corn yield in USA, and was attributed to extensive useof a single genetic male sterility factor
which was unfortunately linked to the disease susceptibility.
Genotyping of cultivars
The repetitive and arbitrary DNA markers are markers of choice in genotyping of cultivars. Microsatellites like (CT)
10, 
(GAA)
5, (AAGG)
4, (AAT)
6(GATA)
4, (CAC)
5and minisatellites have been employed in DNA fingerprinting for the 
detection of genetic variation, cultivar identification and genotyping. This information is useful for quantification of 
genetic diversity, characterization of accessions in plant germplasm collections and taxonomic studies.

14. 09‐May‐17
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Indian scenario for development of molecular markers in crop
improvement programmes
• Agriculture is one of the most important occupations in India with almost55% of the population being dependent
on it. A noteworthy research in conventional breeding for several years has made this country self-sufficient in
many respects. However, the ever-increasing population has alarmed foodsecurity in India and attempts have
been initiated to integrate modern biotechnology tools in conventional breeding to improve the most important
crops such as rice, wheat and legumes.
• Extensive research using DNA markers is in progress in many institutionsall over India. Markers tagged and
mapped with specific genes have been identified
•such examples-include resistance genes for blas and gall midge using RFLP- and PCR-basedapproaches in
rice.
• Similarly, in wheat, leaf rust resistance geneLR28 , and -harvest sprout tolerance gene have been tagged. QTLs
such as protein content in wheat and heterosis in rice
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have also been identified.
While efforts for tagging genes providing resistance to BPH, WBPH, sheathrot and drought are going on,
many attempts are also being made towards pyramiding different resistance genes for a specific disease or pest
attack like blast, bacterial blight, gall midge, BPH, WBPH, etc. in rice inorder to increase the field life of the crop.
• Germplasm analysis to study geneticdiversity is another important areain which a lot of efforts have been put in.
Fingerprinting of crops like rice
,
wheat, chickpea, pigeonpea, pearlmillet etc. is being carried out extensively. This
information has potential in strategic planning of future breeding towards crop sustainability in India.
• Apart from use of molecular markers in crop plants, efforts are also underway in other horticultural plants. Early
identification of sex in dioecious papaya using molecular marker is one such example.