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Assignment:DNA polymorphism and its importance

Submitted to:Dr. Ashrad javeed

Submitted by:Wajiha iram

Institute of Mycology and Plant Pathology

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DNA Polymorphism

and its Importance

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INTRODUCTION

Genetic polymorphism is the existence of variants with respect to a gene locus

(alleles), a chromosome structure (e.g., size of centromeric heterochomatin), a gene

product (variants in enzymatic activity or binding affinity), or a phenotype. The term

DNA polymorphism refers to a wide range of variations in nucleotide repeats, or single

nucleotide variants and they provide the basis for direct physical analysis of genotype

using molecular methods.

Some of the DNA sequence polymorphisms, like our spore color example, occur

within functional genes. DNA polymorphisms, however, have two advantages over

conventional functional mutations.

The first is that the sequence difference is detected directly and no functional

phenotype need ever be associated with that sequence. DNA polymorphisms that

have no known function are called anonymous loci.

The second advantage of DNA polymorphisms is that they occur in a genome at

a very high frequency. One reason for their high frequency is that although

functional gene mutations are by definition limited to coding region. DNA

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polymorphism occurs to any DNA sequence, whether it contributes to a coding

region or not.

DNA polymorphisms that do not affect a phenotype are not subject to selection

pressure, so the majority of DNA variation is located in non-coding regions. Up to 30%

of fungal nuclear DNA is comprised of non-coding sequences (i.e., spacers, introns, and

various sorts of repeated sequence). Variations in any of these regions are detectable as

DNA polymorphisms, but not detectable at all as functional mutations. As compare to

fungi, in humans and other higher eukaryotes 95% of DNA can be comprised of non-

coding sequence. It contributes to chromosome structure (e.g., the DNA telomeres and

centromeres), otherwise has no known function. Because small fraction of DNA is

involved in functional coding, most of these nucleotide changes do not have any effect on

the phenotype of the organism.

DNA polymorphisms are used in genetic mapping studies to identify DNA

markers that are genetically linked to disease genes in the chromosomes in order to

pinpoint their location. They are also used in DNA typing for identifying individuals,

tracking the course of virus and bacterial epidemics, studying human population history

and improving cultivated plants and domesticated animals, as well as for genetic

monitoring of endangered species and for many other purposes.

Types of DNA polymorphisms:

Essentially seven major types of DNA polymorphisms can be used as genetic markers:

Single nucleotide polymorphism (SNPs): for example, substitutions, deletions,

and insertions

Restriction fragment length polymorphism (RFLPs): representing a subtype of

single nucleotide changes that lead to changes at restriction sites.

Amplified Fragment Length Polymorphisms (AFLPs)

Short tandem repeat polymorphism (STRPs).

Variable number of tandem repeat polymorphism (VNTR).

Interspersed Repetitive DNA Polymorphism.

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Randomly amplified polymorphic DNAs (RAPDs)

Each of these types of DNA polymorphisms has an associated set of methods that has

been developed for their analysis (Table 1).

1- Single nucleotide polymorphism (SNPs):

Polymorphisms corresponding to differences at a single nucleotide position (e.g.,

substitution, deletions, and insertions) are called single base pair polymorphisms or more

recently, single nucleotide polymorphisms.

Figure 1: Single nucleotide polymorphism showing DNA sequence variation that occurs when a single nucleotide (A, T, C or G) in the genetic sequence altered. In order for an altered sequence to be considered a SNP, it must occur in a least 1% of the population.

Most polymorphisms of this type have only two alleles (i.e., they are biallelic) and

thus, they are sometimes referred to as biallelic markers. With only two alleles, the

maximum heterozygosity for each SNP marker is only 50%, making them generally less

informative than STRPs (which typically have multiple alleles and heterozygosities well

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over 70%). In general, maps consisting of SNPs need at least three times more markers

than those containing STRPs at comparable resolution (Kruglyak 1997).

SNP Distribution is not uniform for any of the three categories:

Over a complete genome (1/3 in coding, 2/3 in non-coding).

Over all the chromosomes (fewer SNPs in sex chromosomes).

Over a single chromosome (SNPs often concentrated around a specific location).

Coding Region SNPs:

Types of coding region SNPs

Synonymous: the substitution causes no amino acid change to the protein it

produces. This is also called a silent mutation

Non-Synonymous: the substitution results in an alteration of the encoded amino

acid. A missense mutation changes the protein by causing a change of codon. A

nonsense mutation results in a misplaced termination. One half of all coding

sequence SNPs result in non-synonymous codon changes.

SNPs occur frequently in most genomes and have low mutation rate; features that

make them desirable for use in building comprehensive genetic maps. For example, in the

human genome, a SNP with heterozygosity greater than 30% occurs, on average,

approximately every 1.3kb. The increased production of genomic sequence data in

conjunction with improved methods for analysis are leading to the systematic generation

of genetic maps consisting of SNPs.

Methods for the analysis of SNPs:

A number of different methods are available for the analysis of SNPs (Table 1).

Some can only be used to analyzed known SNPs, whereas others can be used both to

identify previously unknown SNPs and to analyze known SNPs. Almost all techniques

used to identifying unknown single nucleotide variations require gel based

electrophoresis.

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Some exploit differences in the electrophoretic or chromatographic mobility

between DNA variants. Examples include DGGE, SSCP analysis, heteroduplex

analysis, TGGE and denaturing HPLC.

Some are based on chemical or enzymatic cleavage of mismatched heteroduplex

DNA fragments followed by gel electrophoresis to look for changes in fragment

length. Examples include CCM, CDI, and T4 endonulease VII (enzymatic)

cleavage of mismatch.

Other methods use various DNA sequencing based approaches. These include

UNG mediated T scan sequencing and direct DNA sequencing.

In general all techniques that can be used for the analysis of both known and

unknown SNPs are relatively labor intensive and difficult to automate. In addition, gel

Table 1: Method for analyzing DNA polymorphism

Polymorphic type Analysis technique Basic methodology

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SNP or insertion / deletion

Tandem array length

Unknown probably SNPs

Denaturing gradient gel Electrophoresis (DGGE)

Single stranded conformationalPolymorphism (SSCP)

Heteroduplex analysis (HP)

Temperature gradient gel Electrophoresis (TGGE)

Cleavase fragment lengthPolymorphism (CFLP)

Chemical cleavage of mismatch (CCM)

Carbodiimide modification (CDI)

Enzymatic cleavage of mismatch(ECM)

UNG- mediated T scan

Direct sequencing

DNA chip resequencing

Allele specific primer extention(GBA, TDI)

Oligonucleotide ligation assay (OLA, DOL)Taqman- ASO

Restriction fragment length Polymorphism (RFLP)

STRP analysis

Minisatellite (VNTR) analysis

Randomly amplified polymorphic DNAs (RAPDs)

PCR / gel electrophoresis

PCR / gel electrophoresis

PCR / gel electrophoresis

PCR / gel electrophoresis

PCR / cleavase treatment/ gel electrophoresis

PCR / chemical cleavage/ gel electrophoresis

PCR / chemical modification/ gel electrophoresisPCR / enzymatic cleavage/ gel electrophoresis

PCR / UNG treatment/ gel electrophoresis

PCR / sequencing / gel electrophoresis

PCR / hybridization/ fluorescence detectionPCR / sequencing/ ELISA or fluorescence detectionPCR /ligation/ ELISA or fluorescence detectionPCR / nuclease cleavage/ ELISA or fluorescence detection

Restriction enzyme digestion/ southern hybridization or PCR / restriction digestion/ gel electrophoresis

PCR/ gel electrophoresis or primary extension/ mass spectrometery

Restriction enzyme digestion/ southern hybridization

PCR/ gel electrophoresis

based methods are susceptible to problems caused by nonspecific products generated

during PCR.

Methods developed specifically for the analysis of known SNPs generally do not

involve gel electrophoresis. They offer tremendous advantage with respect to the

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throughput and potential automation. Such nongel based approaches combine PCR with

an allelic discrimination reaction followed by product detection.

In general, the allelic discrimination step is based on one of three approaches:

allele specific hybridization

allele specific primer extension with DNA polymerase

allele specific ligation with DNA ligase.

The reaction format largely dictated by the detection scheme used. Solid phase

formats are utilized for colorimetric ELISA detection or fluorescence imaging, whereas

homogeneous formats are used for FRET detection. Examples of detection schemes with

solid phase format include DNA chip resequencing with fluorescence detection allele

specific hybridization, genetic bit analysis with ELISA detection and OLA with ELISA

detection.

Examples of homogeneous detection scheme include the TaqMan-ASO assay (5’

nuclease cleavage of hybridized allele specific probe), the TDI assay and DOL assay, all

with energy transfer detection. Since all these methods have two level of specificity (PCR

followed by an allelic discrimination reaction), they are generally not susceptible to

problems caused by nonspecific PCR products. Additionally, the output from these

assays is either positive or negative and can be readily scored in an automated fashion,

thereby minimizing errors associated with human interpretation of results.

Pattern and distribution of SNP in fungi:

Relatively little is known about the patterns and distributions of SNPs in non-

model organisms, including most fungi. In human pathogenic yeast Candida albicans,

there is an SNP frequency of ~1% per nucleotide (Jones et al., 2004). Similarly, a survey

of seven genomic loci for 84 natural strains of the model yeast Saccharomyces cerevisiae

from Asia has identified a total of 62 SNPs, yielding an SNP frequency of 2.05%per

nucleotide (Ayoub et al., 2006). The SNP frequencies are higher in populations of several

opportunistic pathogenic yeasts, such as Candida parapsilosis (~3.4 %; Fundyga et al.,

2004), and the species complexes of Candida guilliermondii (~6.3 %; Lan & Xu, 2006)

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and Cryptococcus neoformans (~20% per nucleotide; Xu et al., 2000). Analysing more

strains should yield additional SNPs within the sequenced DNA fragments. Nevertheless,

all the abovementioned fungi have SNP frequencies much higher than that in the human

genome, in which SNPs are observed approximately once every 250 bp (Miller et al.,

2005).

SNPs Applications:

Single nucleotide polymorphisms are commonly used in:

Pharmacogenomics

Diagnostic genomics

Functional proteomics

Therapeutic genomics

2- Restriction fragment length polymorphism (RFLPs):

The number and length of DNA fragments resulting from digestion of genomic

DNA with a restriction enzyme vary according to the number of restriction sites along the

DNA molecule and the distance between the restriction sites. Variation in a base

sequence can create or destory a restriction site, giving rise to a detectable variation in the

length of DNA fragment. Such genetic variation is known as restriction fragment length

polymorphism (RFLP) and represents the first type DNA polymorphism that was

identified with Southern blot method.

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This technique has been applied widely to plant pathogens, but has particularly

important in taxonomic studies of fungi. It has been used to differentiate isolates of

Phialophora gregata from different hosts, aggressive and nonaggressive isolates of

Ophiostoma ulmi, and dictinct genetic populations with Clletotrichum gloeosporioides as

well as to clearify taxonomic issues in fungal genera including Verticillium, fusarium,

phytopthora, pythium, sclerotinia, and armillaria.( Rudra P. Singh, 1995).

Figure 2 Restriction Fragment Length Polymorphism (RFLP) resulting from b-globin gene mutation.  In the normal cell, the sequence corresponding to 5th to 7th amino acids of the b-globin peptide is CCTGAGGAG, which can be recognized by the restriction enzyme MstII.  In the sickle cell, one base is mutated from A to T, making the site unrecognizable by MstII.  Thus, MstII will generate 0.2 kb and 1.2 kb fragments in the normal cell, but generate 1.4 kb fragment in the sickle cell.  These different fragments can be detected by southern blotting.

Methods for the analysis of RFLP:

Methods to enrich directly for hybridization probes that detect RFLPs have been

developed that involve :

use of genomic substraction techniques e.g., RDA or “RFLP substraction” .

These approaches could make the development of RFLP based maps more

attractive, especially for use with organisms where no genetic map exists.

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Another approach for efficiently identifying RFLPs involves the use of

hybridization probes specific for moderately repetitive sequences. This strategy

has been used successfully in mice with polymorphic insertion sites of retrovirus

related sequences and in human being with “minisatellite” sequences (Table 1).

In addition to analysis by stranded gel transfer hybridization procedures (i.e.,

Southern analysis), RFLPs can be analyzed by the restriction digestion of a PCR

amplified DNA segment that contains the variably present restriction site. Of

course, this requires some knowledge of the DNA sequence flanking that

restriction site ( at least enough to design a PCR assay that amplifies a DNA

fragment containing the site). Such sequence information is generally not

available for most RFLPs. However, known sequence variants ( e.g, mutations)

often disrupt or create a restriction site, allowing for the development of a

convenient RFLP based assay that involves PCR amplification, restriction

digestion and gel electrophresis (without subsequent Southern blot analysis).

Advantages of RFLPs:

Advantages of RFLPs includes:

the relatively low cost and simple methods associated with both their initial

identification and subsequent analysis

the abilty to use gene containing DNA segments (cDNA or genomic) as RFLP

detecting probes

generation (as a by-product) of single copy probes that are useful for numerous

other purposes.

Disadvantages of RFLPs:

Disadvantages of RFLPs includes:

the difficulting in automating the methods used for either their initial

identification or subsequent analysis

the inherent reliance on DNA probes that must be physically distributed in the

case of a given RFLP ( in contrast to primer sequences that can be electronically

distributed in the case of PCR based genetic markers)

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need for large amounts of genomic DNA for detecting RFLPs by Southern blot

analysis.

3- Amplified fragment length polymorphisms (AFLPs):

Amplified Fragment Length Polymorphisms (AFLPs) are differences in

restriction fragment lengths caused by SNPs or INDELs (Insertion/Deletion) that create

or abolish restriction endonuclease recognition sites. AFLP is a powerful tool for

comparative genome analysis, which is described by Zabeau & Vos in 1993. The

procedure of this technique is divided into three steps:

1. Digestion of total cellular DNA with one or more restriction enzymes and ligation

of restriction half-site specific adaptors to all restriction fragments.

2. Selective amplification of some of these fragments with two PCR primers that

have corresponding adaptor and restriction site specific sequences.

3. Electrophoretic separation and amplicons on a gel matrix, followed by

visualisation of the band pattern

AFLP has originally been presented as a tool for strain typing purposes; it is also

very well suited to simultaneously resolve isolates belonging to different species from

each other. This is relevant in the case where the identification of a microorganism may

be uncertain such as the species from the morphologically very similar. If not properly

identified, use of such typing data could easily lead to false conclusions. In a way, AFLP

can be considered the perfect PCR alternative to DNA-DNA re-association studies.

Classical DNA-DNA re-association studies rely on sequence similarities. If two species

share a certain amount of sequence information, it is to be expected that they will also

share a certain amount of similarity in banding patterns from an AFLP fingerprint. In

fact, this has already been demonstrated for a variety of bacterial and fungal species.

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FIGURE 3 An overview of the AFLP technology

Advantages:

no sequence information is required

the PCR technique is fast

a high multiplex ratio is possible

APPLICATIONS :

Genetic Diversity Analysis

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Variety Identification

Acceleration of Inbred Conversions

Removal of Linkage Drag

Linked Marker assay Development

Seed and Plant Quality Assay Development

Introgression Line Library Construction

AFLPs can also be used to compare the hyper- and hypo-methylated regions of

the genome using methylation sensitive and insensitive restriction enzyme

isoschizomers facilitating the detection of possible epigenetic effects

Genetic Map Construction

4- Short tandem repeat polymorphism (STRPs):

Short tandem repeat polymorphism (STRPs), also called microsatellite market,

consist of a short sequence, typically from one or four nucleotides long that is tandemly

repeated several times, and often characterized by many alleles.

For example (CATG)n in a genomic region and is typically in the non-coding

intron region. By identifying repeats of specific sequences, it is possible to create a

genetic profile of an individual. There are currently over 10,000 published STR

sequences in the human genome. STR analysis has become the prevalent analysis method

for determining genetic profiles in forensic cases.

STRP markers can be detected through the use of restriction endonucleases that

cut on either side of the repeat, followed by Southern blotting and detection with a probe

specific for the repeated sequence. Alternatively, PCR amplification with primers located

just outside of the region containing the repeated sequence can be used to generate

amplification fragments whose lengths reflect the number of repeats.

When tandem repeated sequences are replicated during cell division, the number

of repeats can change. Variation in the number of tandem repeats will result in variable

length DNA fragments that can be detected by Southern blotting or PCR, depending of

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the length of the tandem repeat. If the repetitive sequence involves more than 5 base pairs

(generally around 10 to 15 base pairs), these polymorphisms are called variable number

of tandem repeat (VNTR) and are visualized by Southern blotting. On the other hand, if

the repetitive sequence is shorter, these polymorphisms are called short tandem repeats or

microsatellites and are best visualized by PCR. The most frequents type of microsatellites

are the dinucleotide repeats involving cytosine and adenosine (CA repeats), whose length

vary approximately between 24 base pairs (12 repeats) and 80 base pairs (40 repeat). This

type of polymorphism is illustrated in figure 4.

Figure 4 Short tandem repeat polymorphism

The number of copies of tandem repeats is indicated by the number of boxes and

determines the size of DNA fragment measured between the sites of the two primers. It is

visualized by separating the PCR products by electrophoresis. These short tandem repeat

polymorphisms are abundant, evenly distributed within the genome, and highly

polymorphic, making them the most widely used markers to map genetic diseases and to

study the molecular basis of multifunctional phenotypes

Microsatellites or STR"s are ubiquitously present in the genomes of many fungi

including Aspergillus spp. STR are widely used for genetic mapping and strain typing

Size of DNA fragment (in bp)

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purposes. Bart-Delabesse et al. (1998) reported the first application of microsatellites for

A. fumigatus. These markers were obtained by screening genomic DNA libaries of A.

fumigatus for suitable, microsatellite containing sequences, a process that proved quite

laborious in the pre-genomics era. A panel of 4 dinucleotide repeats was selected that

performed well in comparative genotyping experiments (Lasker 2002). Recently, based

on genomic sequence data that has become available, de Valk et al. (2005) reported a

novel set of 9 tandem repeats for typing A. fumigatus isolates, the so-called STRAf assay

(STR”s of A.fumigatus).

Advantages of STRs over traditional RFLP techniques:

Discrete alleles from STR systems may be obtained due to their smaller size,

which puts them in the size range where DNA fragments differing by a single

base pair in size may be differentiated.

Smaller quantities of DNA, including degraded DNA, may be typed using STRs.

STR processing is rapid and abundant STR are available in the human genome.

Discrete alleles allow digital record of data that in turn allows the automated

analysis through a computer.

5- Variable number of tandem repeat polymorphism (VNTR):

VNTR’s are polymorphisms where a particular base sequence, often less than 20

bp, is repeated, from a few to more than 60, at a specific locus. These can be found on

many chromosomes, and often show variations in length between individuals

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Figure 5 Schematic of a Variable Number of Tandem Repeats in 4 alleles.

VNTR structure and allelic variation

In the schematic above, the rectangular blocks represent each of the repeated

DNA sequences at a particular VNTR location. The repeats are tandem - they are

clustered together and oriented in the same direction. Individual repeats can be removed

from (or added to) the VNTR via recombination or replication errors, leading to alleles

with different numbers of repeats. Flanking the repeats are segments of non-repetitive

sequence (shown here as thin lines), allowing the VNTR blocks to be extracted with

restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain

reaction (PCR) technique and their size determined by gel electrophoresis.

Variable Number of Tandem Repeats (VNTR) polymorphism has been identified

in the ITS1 region of the rDNA in 13 strains belonging to a species of Nodulisporium

producing the novel indole diterpene nodulisporic acid. The number of tandem repeats

found in these isolates varies from 2, 4 or 5 repetitions, and produces three length morphs

in the ITS1 rDNA, spanning a length difference of 217, 308 and 352 nucleotides.

Use of VNTRs in genetic analysis

VNTRs were an important source of RFLP genetic markers used in linkage

analysis (mapping) of genomes.

Now that many genomes have been sequenced, VNTRs have become essential to

forensic crime investigations, via DNA fingerprinting and the CODIS database.

When removed from surrounding DNA by the PCR or RFLP methods, and their

size determined by gel electrophoresis or Southern blotting, they produce a

pattern of bands unique to each individual. When tested with a group of

independent VNTR markers, the likelihood of two unrelated individuals having

the same allelic pattern is extremely improbable.

VNTR analysis is also being used to study genetic diversity and breeding patterns

in populations of wild or domesticated animals.

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6- Interspersed Repetitive DNA Polymorphism:

Interspersed repetitive DNA is a type of polymorphism where a particular base

sequence is repeated throughout the genome (not in tandem, at a single locus). Only one

copy of the sequence is located at any given locus, but that same sequence is observed at

thousands of different loci. These sequences are sometimes referred to as “satellite DNA”

Interspersed repetitive DNA is thought to make up somewhere between 5% to

20% of the human genome. The percentages and specific distribution patterns of these

dispersed sequences is generally conserved within species, but varies between species.

For this reason, interspersed repetitive DNA sequences have been useful in tracing

evolutionary relationships

.

7- Randomly amplified polymorphic DNAs (RAPDs):

RAPDs can be used in a simple, inexpensive, and efficient fashion for

identifying polymorphic markers and building genetic maps. RAPD- based approaches

have been used for identifying polymorphic markers between inbred populations.

Specifically, the use of RAPDs involves low stringency PCR of genomic DNA with a

single, short Oligonucleotide primer. The products generated from different individuals

are then subjected to gel electrophoresis and directly compared, with any reliable

differences in the number and/ or size of products reflecting a polymorphism.

RAPD can be useful for detecting genetic differences within species (Williams et

al., 1990, Parker et al., 1998, Sunnucks 2000). This technique has been used to

investigate intra specific genetic variation in several fungi (e.g., Fegan et al., 1993,

Moore et al., 2001). RAPD markers were used to examine the degree of genetic variation

within the putatively asexual basidiomycete fungus (Lepiotaceae: provisionally named

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Leucoagaricus gongylophorus) associated with the leaf-cutting ant species Atta

cephalotes.

RAPD markers have also been useful in other investigations of genetic variation

among geographically distant populations of fungi. Li et al., (2001) compared RAPD

markers in populations of western gall rust fungus (Endocronartium harknesii) collected

across western and central Canada from two host pine species. Most of the genetic

variation in E. harknesii occurred between the two host species, but within hosts there

was more variation among geographically widespread locations than within locations.

Advantages of RAPD:

The main advantages of RAPDs are

The low cost (corresponding to a few short oligonucleotides and several PCR

assays).

Potential for rapid identification of numerous polymorphisms.

Disadvantages of RAPD:

However the disadvantages are significant and include

The lack of a ‘molecular handle’ (i.e., the ability to directly access) on any of the

resulting polymorphism

The lack of predictability,

In some cases, the lack of reproducibility of low stringency PCR

The fact that the marker are scored dominantly.

This last feature reduces the amount of genetic information that can be readily

obtained. Nevertheless, the use of RAPDs in some settings should be considered, since

there is no more rapid and inexpensive methods for developing genetic markers or maps

for an organism.

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Importance of DNA polymorphism:

1- Human population history

DNA polymorphisms provide important information in anthropology to

reconstruct the evolutionary origin, global expansion, and diversification of the

human population.

2- Improvement of domesticated plants and animals

Plant and animal breeders have turned to DNA polymorphisms as genetic markers

in pedigree studies to identify, by genetic mapping; genes that are associated with

favorable traits in order to incorporate these genes into currently used varieties of

plants and breeds of animals.

3- History of domestication

Plants and animal breeders also study genetic polymorphism to identify the wild

ancestors of cultivated plants and domesticated animals, as well as to infer the

practices of artificial selection that led to genetic changes in these species during

domestication.

4- DNA polymorphism as ecological indicators

DNA polymorphisms are being evaluated as biological indicators of genetic

diversity in key indicator species present in biological communities exposed to

chemical, biological, or physical stress. They are also used to monitor genetic

diversity in endangered species and species bred in captivity.

5- Evolutionary genetics

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DNA polymorphism are studied in an effort to describe the patterns in which

different types of genetic variation occur throughout the genome, to infer the

evolutionary mechanisms by which genetic variation is maintained, and to illuminate

the process by which genetic polymorphism within species become transformed into

genetic differences between species.

6- Population studies

Population ecologists employ DNA polymorphisms to assess the level of genetic

variation in diverse population of organisms that differ in genetic organization

(prokaryotes, eukaryotes, organelles), population size, breeding structure, or life-

history characters, and they use genetic polymorphism within subpopulations of a

species as indicators of population history, patterns of migration, and so forth.

7- Evolutionary relationship among species

Differences in DNA sequences between species is the basis of molecular

phylogentics, in which the sequences are analyzed to determine the ancestral history

(phylogeny) of the species and to trace the origin of morphological, behavioral, and

other types of adaptations that have arisen in the course of evolution.

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