Assignment:

DNA polymorphism and its importance

Submitted to:
Dr. Ashrad javeed

Submitted by:
Wajiha iram

Institute of Mycology and Plant Pathology

 DNA
Polymorphism
and its
Importance
 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
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.

• 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
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.

• 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

SNP or insertion / Denaturing gradient gel
deletion Electrophoresis (DGGE)
Single stranded conformational
Polymorphism (SSCP)
Heteroduplex analysis (HP)
Temperature gradient gel
Electrophoresis (TGGE)
Cleavase fragment length
Polymorphism (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)
Tandem array STRP analysis
length
Minisatellite (VNTR) analysis
Unknown probably Randomly amplified polymorphic
SNPs DNAs (RAPDs)
based methods are susceptible to problems caused by nonspecific products generated
during PCR.
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
electrophoresis
PCR / enzymatic cleavage/ gel
electrophoresis
PCR / UNG treatment/ gel electrophoresis
PCR / sequencing / gel electrophoresis
PCR / hybridization/ fluorescence detection
PCR / sequencing/ ELISA or fluorescence
detection
PCR /ligation/ ELISA or fluorescence
detection
PCR / 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
 Methods developed specifically for the analysis of known SNPs generally do not
involve gel electrophoresis. They offer tremendous advantage with respect to the
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)
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.
 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.
 • 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)
• 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.

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

• 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
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.

Size of DNA
fragment (in bp)

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
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
 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.

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
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.

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

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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