Molecular markers for measuring genetic diversity Introduction: The molecular basis of the essential biological phenomena in plants is crucial for the effective conservation, management, and efficient utilization of plant genetic resources (PGR). Determining genetic diversity can be based on morphological, biochemical, and molecular types of information. However, molecular markers have advantages over other kinds, where they show genetic differences on a more detailed level without interferences from environmental factors, and where they involve techniques that provide fast results detailing genetic diversity Comparison of different methods Morphological characterization does not require expensive technology but large tracts of land are often required for these experiments, making it possibly more expensive than molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely, this allows assessment of diversity in the presence of environmental variation. Biochemical analysis is based on the separation of proteins into specific banding patterns. It is a fast method which requires only small amounts of biological material. However, only a limited number of enzymes are available and thus, the resolution of diversity is limited. Molecular analyses comprise a large variety of DNA molecular markers, which can be employed for analysis of variation. Different markers have different genetic qualities (they can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain expressed or non-expressed sequences, etc.). Genetic marker The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic markers for Drosophila melanogaster led to the founding of the theory of genetic linkage. A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be used in the laboratory to tell apart cells, individuals, populations, or species. The use of genetic markers begins with extracting proteins or chemicals (for biochemical markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage, pollen, sometimes woody tissues). Molecular markers In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is associated with a certain location within the genome. Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion, duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e. dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant markers are more informative than the

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Molecular markers for measuring genetic
diversity
Introduction:
The molecular basis of the essential biological phenomena in plants is crucial for the effective
conservation, management, and efficient utilization of plant genetic resources (PGR).
Determining genetic diversity can be based on morphological, biochemical, and molecular types
of information. However, molecular markers have advantages over other kinds, where they show
genetic differences on a more detailed level without interferences from environmental factors,
and where they involve techniques that provide fast results detailing genetic diversity
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Comparison of different methods
 Morphological characterization does not require expensive technology but large tracts of
land are often required for these experiments, making it possibly more expensive than
molecular assessment. These traits are often susceptible to phenotypic plasticity; conversely,
this allows assessment of diversity in the presence of environmental variation.
 Biochemical analysis is based on the separation of proteins into specific banding patterns. It
is a fast method which requires only small amounts of biological material. However, only a
limited number of enzymes are available and thus, the resolution of diversity is limited.
 Molecular analyses comprise a large variety of DNA molecular markers, which can be
employed for analysis of variation. Different markers have different genetic qualities (they
can be dominant or co-dominant, can amplify anonymous or characterized loci, can contain
expressed or non-expressed sequences, etc.).
Genetic marker
The concept of genetic markers is not a new one; in the nineteenth century, Gregor Mendel
employed phenotype-based genetic markers in his experiments. Later, phenotype-based genetic
markers for Drosophila melanogaster led to the founding of the theory of genetic linkage.
A genetic marker is an easily identifiable piece of genetic material, usually DNA that can be
used in the laboratory to tell apart cells, individuals, populations, or species.
The use of genetic markers begins with extracting proteins or chemicals (for biochemical
markers) or DNA (for molecular markers) from tissues of the plant (for example, seeds, foliage,
pollen, sometimes woody tissues).
Molecular markers
In genetics, a molecular marker (identified as genetic marker) is a fragment of DNA that is

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associated with a certain location within the genome.
Molecular markers which detect variation at the DNA level such as nucleotide changes: deletion,
duplication, inversion and/or insertion. Markers can exhibit two modes of inheritance, i.e.
dominant/recessive or co-dominant. If the genetic pattern of homozygotes can be distinguished
from that of heterozygotes, then a marker is said to be co-dominant. Generally co-dominant
markers are more informative than the dominant markers.
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Characteristics of molecular marker
 Be polymorphic and evenly distributed throughout the genome.
 Provide adequate resolution of genetic differences.
 Generate multiple, independent and reliable markers.
 Be simple, quick and inexpensive.
 Need small amounts of tissue and DNA samples.
 Link to distinct phenotypes.
 Require no prior Diversity information about the genome of an organism.
Advantages of molecular marker
 Being applicable to any part of the genome (introns, exons and regulation regions).
 Not possessing pleiotrophic or epistatic effects.
 Being able to distinguish polymorphisms which not produce phenotypic variation and
finally.
 Being some of them co-dominant.
Types of molecular markers
Basic marker techniques can be classified into two categories:
(1) non-PCR-based techniques or hybridization based techniques.
(2) PCR-based techniques.
1. Randomly Amplified Polymorphic DNA (RAPD)
RAPD was the first PCR based molecular marker technique developed and it is by far the
simplest Short PCR primers (approximately 10 bases) are randomly and arbitrarily 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 products are usually visualized on agarose gels stained with ethedium bromide.

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RAPD markers are easily developed and because they are based on PCR amplification followed
by agarose gel electrophoresis, they are quickly and readily detected. RAPD technique was used
extensively in studying genetic diversity between plant species. For example, it was used to
study genetic structure and diversity among and between six populations of Capparis deciduas in
Saudi Arabia.
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2. Amplified Fragment Length Polymorphisms (AFLP)
Amplified Fragment Length Polymorphisms (AFLP) based genomic DNA fingerprinting is a
technique used to detect DNA polymorphism. AFLP is a polymerase chain reaction (PCR) based
technique, has been reliably used for determining genetic diversity and phylogenetic relationship
between closely related genotypes. AFLP markers are generally dominant and do not require
prior knowledge of the genomic composition. AFLPs are produced in great numbers and are
reproducible
The AFLP is applicable to all species giving very reproducible results. It was also used in
microbial population: in studying genetic diversity of human pathogenic bacteria. In that regards
it has the advantage of the extensive coverage of the genome under study. In addition the
complexity of the bands can be reduced by adding selective bases to the primers during PCR
amplification. It was also used in studying genetic diversity of human pathogenic bacteria the
completion of the genome sequencing of E. coli, it was possible to predict the band pattern of the
AFLP analysis of E. coli. This indicates the power of this technique. In higher plants AFLP was
used in variety of applications which includes examining genetic relationship between species
investigating genetic structure of gene pool and assessment of genetic differentiation among
populations
3. Single nucleotide polymorphism SNP’s
Single nucleotide polymorphism SNP’s, represent sites in the genome where DNA sequence
differs by a single base when two or more individuals are compared. They may be individually
responsible for specific traits or phenotypes, or may represent neutral variation that is useful for
evaluating diversity in the context of evolution. SNPs are the most widespread type of sequence
variation in genomes discovered so far. About 90% of sequence variants in humans are
differences in single bases of DNA
Several disciplines such as population ecology and conservation and evolutionary genetics are
benefitting from SNPs as genetic markers. There is widespread interest in finding SNP’s because
they are numerous, more stable, potentially easier to score than the microsatellite repeats
currently been used in gene mapping in human. Within coding regions there are on average four
SNPs per gene with a frequency above 1%. About half of these cause amino acid substitutions:
termed non-synonymous SNPs

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Because of the importance of the SNP’s in the discovery of DNA sequence variants, the National
Human Genome Research Institute (NHGRI) of NIH along with the Center for Disease Control
and Prevention and several individual investigators have assembled a DNA Polymorphism
Discovery Resource of samples from 450 U.S. residents This DNA variant discovery will help in
finding SNP’s that are deleterious to gene function or likely to be disease associated.
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4. Microsatellite-Based Marker Technique
Microsatellites or Simple Sequence Repeats (SSR) are sets repeated sequences found within
eukaryotic These consist of sequences of repetitions, comprising basic short motifs generally
between 2 and 6 base-pairs long. Polymorphisms associated with a specific locus are due to the
variation in length of the microsatellite, which in turn depends on the number of repetitions of
the basic motif. Variations in the number of tandemly repeated units are mainly due to strand
slippage during DNA replication where the repeats allow matching via excision or addition of
repeats. As slippage in replication is more likely than point mutations, microsatellite loci tend to
be hypervariable.
Microsatellite assays show extensive inter-individual length polymorphisms during PCR analysis
of unique loci using discriminatory primers sets. Microsatellites are highly popular genetic
markers as they possess: co-dominant inheritance, high abundance, enormous extent of allelic
diversity, ease of assessing SSR size variation through PCR with pairs of flanking primers and
high reproducibility. However, the development of microsatellites requires extensive knowledge
of DNA sequences, and sometimes they underestimate genetic structure measurements, hence
they have been developed primarily for agricultural species, rather than wild species. Initial
approaches were principally based on hybridization techniques, whilst more recent techniques
are based on PCR.
Major molecular markers based on assessment of variability generated by microsatellites
sequences are: STMSs (Sequence Tagged Microsatellite Site), SSLPs (Simple Sequence Length
Polymorphism), SNPs (Single Nucleotide Polymorphisms), SCARs (Sequence Characterized
Amplified Region) and CAPS (Cleaved Amplified Polymorphic Sequences)
5. Restriction-Hybridization Techniques (Non-PCR-Based)
Molecular markers based on restriction-hybridization techniques were employed relatively
early in the field of plant studies and combined the use of restriction endonucleases and the
hybridization method. Restriction endonucleases are bacterial enzymes able to cut DNA,
identifying specific palindrome sequences and producing polynucleotidic fragments with
variable dimensions. Any changes within sequences (i.e., point mutations), mutations between
two sites (i.e., deletions and translocations), or mutations within the enzyme site, can generate
variations in the length of restriction fragment obtained after enzymatic digestion.

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RFLP and Variable Numbers of Tandem Repeats (VNTRs) markers are examples of molecular
markers based on restriction-hybridization techniques. In RFLP, DNA polymorphism is detected
by hybridizing a chemically-labelled DNA probe to a Southern blot of DNA digested by
restriction endonucleases, resulting in differential DNA fragment profile. The RFLP markers are
relatively highly polymorphic, codominantly inherited, and highly replicable and allow the
simultaneously screening of numerous samples. DNA blots can be analyzed repeatedly by
stripping and reprobing (usually eight to ten times) with different RFLP probes. Nevertheless,
this technique is not very widely used as it is time-consuming, involves expensive and
radioactive/toxic reagents and requires large quantities of high quality genomic DNA. Moreover,
the prerequisite of prior sequence information for probe construction contributes to the
complexity of the methodology. These limitations led to the development of a new set of less
technically complex methods known as PCR-based techniques.
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