Molecular Marker: Study Notes

Molecular Markers:
A molecular marker is a DNA sequence in the genome which can be
located and identified. As a result of genetic alterations (mutations,
insertions, deletions), the base composition at a particular location of
the genome may be different in different plants.

These differences, collectively called as polymorphisms can be mapped

and identified. Plant breeders always prefer to detect the gene as the
molecular marker, although this is not always possible. The alternative
is to have markers which are closely associated with genes and
inherited together.

The molecular markers are highly reliable and advantageous

in plant breeding programmes:
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i. Molecular markers provide a true representations of the genetic

makeup at the DNA level.

ii. They are consistent and not affected by environmental factors.

iii. Molecular markers can be detected much before development of

plants occur.

iv. A large number of markers can be generated as per the needs.

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Basic principle of molecular marker detection:

Let us assume that there are two plants of the same species—one with
disease sensitivity and the other with disease resistance. If there is
DNA marker that can identify these two alleles, then the genome can
be extracted, digested by restriction enzymes, and separated by gel
electrophoresis. The DNA fragments can be detected by their
separation.For instance, the disease resistant plant may have a shorter
DNA fragment while the disease — sensitive plant may have a longer
DNA fragment (Fig. 53.1).

Molecular markers are of two types:

1. Based on nucleic acid (DNA) hybridization (non-PCR based
approaches).

2. Based on PCR amplification (PCR-based approaches).

Markers Based On DNA Hybridization:

The DNA piece can be cloned, and allowed to hybridize with the
genomic DNA which can be detected. Marker-based DNA
hybridization is widely used. The major limitation of this approach is
that it requires large quantities of DNA and the use of radioactivity
(labeled probes).

Restriction fragment length polymorphism (RFLP):

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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. 53.2, and schematically depicted in Fig.
53.3.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
(Fig. 53.2).
Based on the presence of restriction sites, DNA fragments of different
lengths can be generated by using different restriction enzymes. In the
Fig. 53.3, two DNA molecules from two plants (A and B) are shown. In
plant A, a mutations has occurred leading to the loss of restriction site
that can be digested by EcoRI.

The result is that when the DNA molecules are digested by the enzyme
Hindlll, there is no difference in the DNA fragments separated.
However, with the enzyme EcoRI, plant A DNA molecules is not
digested while plant B DNA molecule is digested. This results in a
polymorphic pattern of separation.

Markers Based on PCR Amplification:

Polymerase chain reaction (PCR) is a novel technique for the
amplification of selected regions of DNA .The advantage with PCR is
that even a minute quantity of DNA can be amplified. Thus, PCR-
based molecular markers require only a small quantity of DNA to start
with.

PCR-based markers may be divided into two types:

1. Locus non-specific markers e.g. random amplified polymorphic
DNA (RAPD); amplified fragment length polymorphism (AFLP).

2. Locus specific markers e.g. simple sequence repeats (SSR); single

nucleotide polymorphism (SNP).

Random amplified polymorphic DNA (RAPD) markers:

RAPD is a molecular marker based on PCR amplification. An outline
of RAPD is depicted in Fig. 53.4. 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.
Based on the nucleotide alterations in the genome, the polymorphisms
of amplified DNA sequences differ which can be identified as bends on
gel electrophoresis. Genomic DNA from two different plants often
results in different amplification patterns i.e. RAPDs. This is based on
the fact that a particular fragment of DNA may be generated from one
individual, and not from others. This represents polymorphism and
can be used as a molecular marker of a particular species.

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 (Fig. 53.5).
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.
AFLP analysis is tedious and requires the involvement of skilled
technical personnel. Hence some people are not in favour of this
technique. In recent years, commercial kits are made available for
AFLP analysis. AFLP is very sensitive and reproducible. It does not
require prior knowledge of sequence information. By AFLP, a large
number of polymorphic bands can be produced and detected.

Sequence tagged sites (STS):

Sequence tagged sites represent unique simple copy segments of
genomes, whose DNA sequences are known, and which can be
amplified by using PCR. STS markers are based on the polymorphism
of simple nucleotide repeats e.g. (GA)n, (GT)n, (CAA)n etc. on the
genome. STS have been recently developed in plants. When the STS
loci contain simple sequence length polymorphisms (SSLPs), they are
highly valuable as molecular markers. STS loci have been analysed and
studied in a number of plant species.
Microsatellites:
Microsatellites are the tandemly repeated multi-copies of mono-, di-,
tri- and tetra nucleotide motifs. In some instances, the flanking
sequence of the repeat sequences may be unique. Primers can be
designed for such flanking sequences to detect the sequence tagged
microsatellites (STMS). This can be done by PCR.

Sequence characterized amplified regions (SCARs):

SCARs are the modified forms of STS markers. They are developed by
PCR primer that are made for the ends of RAPD fragment. The STS-
converted RAPD markers are sometimes referred to as SCARs. SCARs
are useful for the rapid development of STS markers.

Molecular Marker Assisted Selection:

Selection of the desired traits and improvement of crops has been a
part of the conventional breeding programmes. This is predominantly
based on the identification of phenotypes. It is now an accepted fact
that the phenotypes do not necessarily represent the genotypes. Many
a times the environment may mark the genotype. Thus, the plant’s
genetic potential is not truly reflected in the phenotypic expression for
various reasons.

The molecular marker assisted selection is based on the identification

of DNA markers that link/ represent the plant traits. These traits
include resistance to pathogens and insects, tolerance to abiotic
stresses, and various other qualitative and quantitative traits. The
advantage with a molecular marker is that a plant breeder can select a
suitable marker for the desired trait which can be detected well in
advance. Accordingly, breeding programmes can be planned.

The following are the major requirements for the molecular

marked selection in plant breeding:
i. The marker should be closely linked with the desired trait.

ii. The marker screening methods must be efficient, reproducible and

easy to carry out.

iii. The analysis should be economical.

Molecular Breeding:
With rapid progress in molecular biology and genetic engineering,
there is now a possibility of improving the crop plants with respect to
yield and quality. The term molecular breeding is frequently used to
represent the breeding methods that are coupled with genetic
engineering techniques.

Improved agriculture to meet the food demands of the world is a high

priority area. For several years, the conventional plant breeding
programmes (although time consuming) have certainly helped to
improve grain yield and cereal production.

The development of dwarf and semi-dwarf varieties of rice and wheat

have been responsible for the ‘Green Revolution’, which has helped to
feed millions of poverty-stricken people around the world. Many
developments on the agriculture front are expected in the coming
years as a result of molecular breeding.
Linkage analysis:
Linkage analysis basically deals with studies to correlate the link
between the molecular marker and a desired trait. This is an
important aspect of molecular breeding programmes. Linkage analysis
has to be carried out among the populations of several generations to
establish the appropriate linkage. In the earlier years, linkage analysis
was carried out by use of isoenzymes and the associated
polymorphisms. Molecular markers are now being used. The
techniques employed for this purpose have already been described.

Quantitative Trait Loci:

These are many characteristics controlled by several genes in a
complex manner. Some good examples are growth habit, yield,
adaptability to environment, and disease resistance. These are
referred to as quantitative traits. The locations on the chromosomes
for these genes are regarded as quantitative trait loci (QTL).

The major problem, the plant breeder faces is how to improve the a
complex character controlled by many genes. It is not an easy job to
manipulate multiple genes in genetic engineering. Therefore, it is a
very difficult and time consuming process. For instance, development
of Golden Rice (with enriched pro-vitamin A) involving the insertion
of just three genes took about seven years.

Arid and Semi-Arid Plant Biotechnology:

The terms arid zone is used to refer to harsh environmental conditions
with extreme heat and cold. The fields have limited water and
minerals. It is different task to grow plants and achieve good crop yield
in arid zones. Semi-arid regions are characterized by unpredictable
weather, inconsistent rainfall, long dry seasons, and poor nutrients in
the soil.

Most parts of India and many other developing countries (Africa,

Latin America, and Southeast Asia) have semi- arid regions. Crops like
sorghum, millet, groundnut and cowpea are mostly grown in semi-arid
tropics. Besides unpredictable weather, biotic and abiotic stresses
contribute to crop loss in these areas.

The biotechnological approaches for the breeding

programmes in the semi-arid regions should cover the
following areas:
i. Development of crops that are tolerant to drought and salinity.

ii. Improvements to withstand various biotic and abiotic stresses.

iii. Micro-propagation techniques to spread economically important

plants which can withstand harsh environmental conditions.

Some success has been achieved in improving sorghum, millet and

legume crops that are grown in semi-arid regions. Genetic
transformation in sorghum was possible by using micro projectile
method.

Greenhouse and Green-home Technology:

Greenhouse literally means a building made up of glass to grow plants.
Green houses are required to grow regenerated plants for further
propagation and for growing plants to maturity. Greenhouses are the
intermediary stages involving the transitional step between the plant
cultures and plant fields. The purpose of greenhouses is to acclimatize
and test the plants before they are released into the natural
environment.

The plants are grown in greenhouse to develop adequate root systems

and leaves so as to withstand the field environment. The greenhouses
are normally equipped with cooling systems to control temperature.
Greenhouses have chambers fitted with artificial lights. It is possible
to subject the plants to different lighting profiles. In recent years many
improvements have been made in the development of more suitable
greenhouses. These include the parameters such as soil, and humidity.

The major limitation of greenhouse technology is an increase in

CO2production that in turn increases temperature. Some approaches
are available to control temperature. Green home technology is a
recent development. In this case, temperature is controlled by using
minimum energy.

Applications of AFLP Marker |

Genetics
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In this article we will discuss about the applications of AFLP markers

in plants. Also learn about its demerits.

AFLP is primarily used in genetic mapping. Several economically

important cereal crops such as rice, barley and wheat have been
mapped by AFLP. The AFLP markers, which are produced by different
combinations of restriction enzymes, are distributed throughout the
genome. Evidence shows that AFLP marker lie outside regions that are
occupied with RFLP. In barley, AFLP markers are located on the long
and short arm of all seven chromosomes.

These AFLP markers exhibit strong relation between the number of

markers per chromosome and length of the chromosome. Similarly, in
rice, cross between Indica X Japonica revealed that 50 AFLP markers
were located on every chromosome except in small chromosome 12.
These polymorphic loci distributed throughout the genome of this
species can be illustrated by AFLP technique.

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The existing barley map developed by RFLP comprises of 157 RFLP

loci has been increased by adding 118 AFLP markers. The total map
length was increased by 71% mainly attributed to gap filling, terminal
extension and general expansion.
The level of polymorphism detected in barley by AFLP can range from
12.2% (between procter × Ny dinka) to 29.0% (between L94 × voda).
However, detection level of this polymorphic range is comparatively
less than that of used by other mapping technique like RFLP.

Increasing AFLP is used for several applications to assist the rapid

isolation and characterization of target genes.

Breeding for resistance is an important programme in cereal research.

AFLP technique identified several markers closely linked to barley Mlo
resistance gene. AFLP based fine mapping of the resistance gene locus
to be delimited to 30 kb.

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i. AFLP is used to screen large number of polymorphism. It is possible

to saturate specific regions of the genome for map-based cloning of
target genes.

ii. AFLP has also been used in phylogenetic studies and distinguishing
feature between varieties. In case of barley studies, varieties are
grouped according to their salt tolerance and area of origin by
genotyping with AFLP. Determination of ancestral origin of wheat
variety using number of AFLP analysis which had previously not been
possible using other molecular techniques due to the low genetic
diversity of races.

iii. AFLP is also used to screen pools of plasmid DNA from several
clones, enabling rapid isolation of genes tightly linked to markers.

iv. AFLP has recently been applied to the analysis of quantitative traits
in barley and rice.

v. RFLP can be used to score semi-dominant markers. This was

possible due to development of new software for image analysis of
fluorescent PCR products developed by key gene. This was probably
developed for use with AFLP and enable AFLP to score semi-dominant
marker.

vi. AFLP is rapidly becomes preferred molecular technique for several

different investigation particularly in many areas of research.

Demerits of AFLP:
i. Not reliable to convert AFLP into SCAR

ii. Null allele cannot be detected

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iii. Proprietary technique

iv. Require high amount of DNA than required in RAPD.

v. Relatively expensive technique owing to requirement of silver

staining and radio or non-radiolabelling