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Vol XXIV, No. 8, Special Issue, 2016

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Setti and Bencheikh Transylvanian Review: Vol XXIV, No. 8, Special Issue, 2016

Applications of Nuclear Ribosomal Internal

Transcribed Spacer (ITS) as Molecular Markers for
Plant Pathogenic Fungus Population Studies
*
Setti B. and Bencheikh M.
*
Laboratoire de Phytopathologie, Institut des Sciences Agronomiques Universit de Chlef, Algrie.

Abstract

Recent advances in molecular technology have been widely applied in many fields in plant pathology including fungal
identification, taxonomics and systemratic studies. These have led to the emergence of internal transcribed spacers as
powerful molecular markers for the study of the plant pathogen populations.The ITS regions occur in large tandem arrays
inside the ribosomal nuclear DNA which include the 18S, 5.8S and the 28S genes. The internal transcribed spacer region of
fungal ribosomal DNA are highly variable sequence. They are also distributed throughout the eukaryotic genome and are
detected by polymerase chain reaction using universal primers. These features make them a marker of choice of numerous
applications including genetic diversity, population structure, phylogenetic relationships, strain identification and
fingerprinting. On the other hand, a computational approach to ITS mining is becoming more popular with the increasing of the
freely databases as well as the number of bioinformatics pipelines. This great popularity of the computational method has led
to the construction of huge numbers of ITS databases for both prokaryotes and eukaryotes organisms including
phytopathogenic microorganisms. The purpose of this review is to present the most relevant published reports with emphasis
on the use of the ITS markers for the population pathogen genetics. Among the aspects reviewed in this paper includes the
distribution and mutations of ITS regions, the main method used for ITSs detection and genotyping and the in silico mining of
ITSs through the publicly databases. Thereafter, we present their various applications in fungal populations studies.

Keywords: Internal transcribed spacer, ITS1, ITS2, population genetics, plant pathogen, fungi, molecular markers.

*
Corresponding author: Laboratoire de phytopathologie, institut des sciences agronomiques universit de chlef, Algrie.
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Setti and Bencheikh
Introduction
In the last two decades, DNA marker technologies
have been revolutionized the plant pathogen genomic
analysis and have been extensively employed in many
fields of molecular plant pathology. Molecular markers
offer also the possibility of faster and accurate
identification and early detection of plant pathogen (Bridge
et al., 2003; Schaad et al., 2003). On the other hand,
molecular techniques provide an alternative methods to
elucidate molecular evolution, population genetics
including the diversity analysis, populations structures
and the population dynamics of plant pathogens (Schaad et
al., 2003). For fungal plant pathogens, the most widely used
molecular markers gene and the most extensively
sequenced region is the internal transcribed spacers
(ITSs). The ITS plays a role in ribosomal maturation of
small and large subunit rRNA (Joseph et al., 1999; Muster
et al., 1990). This region lies between the 18S small subunit
(SSU) and the 28S large subunit(LSU) ribosomal RNA
(rRNA) genes and contains two variable non coding regions
ITS1 and ITS2 separated by 5.8S rRNA gene (Maleszka and
Clark-Walker, 1990; Ganley and Kobayashi, 2007; White et
al., 2013). The ITS spacers regions exist in several
hundreds copies in most fungi and are located in one or
several loci destributed in one or several chromosomes
(Hillis and Dixon,1991; Baldwin et al., 1992; White et al.,
2013).
The advantages of ITSs over the others markers are
due to their highly multiallelic characteristics, their
abundance, reproducibility, inheritance and the rapidity of
their detection based on polymerase chain reaction (PCR)
(Kong et al., 2003 ; Kong et al., 2004). Which make them a
powerful tool and the marker of choice for great numbers
of application in the field of fungal biology covering area of
species identification, fingerprinting, genetic mapping,
Fig. 1: General structure of the rDNA gene in fungal genome with primers ITS1 and ITS4 localization (arrows).
One of the remarkable properties, is that the nuclear
ribosomal genes can be highly homogenous due to the
concerted evolution through number of mechanisms
including gene conversion, unequal crossing over as well
as insertion or deletion of the sequence arrays and
replication slippage (Baldwin et al., 1995; Li, 1997).This
type of unequal crossing over is driven by recombination
among tandem repeats either within chromosome or
between chromosomes (Walsh, 1987; Baldwin et al., 1995;
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Transylvanian Review: Vol XXIV, No. 8, Special Issue, 2016
genetic diversity, population structure and phylogeny
(Ward et al., 1994; Ward and Adams, 1998 ; Henry et al.,
2000; Hebert et al., 2003; Blackwell, 2011). In this article,
we propose to review the method used for development
and detection of ITSs, the major software tools and current
publicly available database and Web services dedicated for
ITSs mining and finally provide an overview of their major
applications in the field of plant pathology.
Distribution and Variability of Internal
Transcribed Spacer (ITSs) Regions.
In eukaryotic organisms, the ribosomal DNA are
highly structured in cluster present in both the nucleus
and the mitochnondria. It is organized in a large arrays of
repetitive transcriptional units, two intranscribed spacers
ITS1 and ITS2 and two external spacer sequences. The
eukaryotic nuclear ribosomal DNA(nrDNA) are tanscribed
as unit by RNA polymerase I into a single precursor
molecule (35-45S preRNA). The product of the polymerase
I undergoes a series of processes in the nucleus where
ITS1 and ITS2 where removed. In fungi, rDNA consists of
ribosomal subunits genes that are separated by internal
transcribed spacers (ITS). This fungal ITS region are
structured in tandem array that may contains 40 to 200
copies (Maleszka and Clark-Walker, 1990; Ganley and
Kobayashi, 2007). Furthermore, the length of fungal ITS
region may vary between 400 to 700bp which are located in
one or several loci and distributed accross one or more
chromosome location. The internal transcribed spacers,
ITS1 and ITS2 lies between the highly conserved 18S small
unit (SSU) and the large unit (LSU) separeted by the 5.8S
ribosomal subunits and is usually amplified by the
universal primer pair ITS1 and ITS4 (Fig. 1) (Hillis and
Dixon, 1991; Baldwin et al., 1992; White et al., 2013).
Li, 1997). The ITS variation show generally a faster
evolutionnary divergence than the coding sequences and
may vary among species and population (Arnheim et al.,
1982; Dover, 1989; Li, 1997).
ITSs Detection and Genotyping Technologies
Several methods were developed recently for
detection and genotyping the internal transcribed spacers.
These methods vary in terms of sensitivity, cost, accuracy
Setti and Bencheikh
and throughput. We try in the next chapter, to describe
briefly the principle of the major and most popular
technologies for ITSs analysis that would be ideal for
population genetic studies. This include the Single
Stranded Conformation Polymorphism (SSCP)(Kong et al.,
2003; Kong et al., 2004), PCR-restriction fragment length
polymorphism (PCR-RFLP)(Gomes et al., 2002), matrix
assisted laser desorption ionization time of flight
(MALDI-TOF)(Chalupov, et al., 2013) and the
pyrosequencing ((Peng et al., 2015).
Single Stranded Conformation Polymorphism
(SSCP)
Single Stranded Conformation Polymorphism (SSCP)
is simple technique that has been used successfully for the
detection of mutation in a nucleotide sequence and thus is
considered as useful tool for detection of internal
transcribed spacers ITS1 and ITS2. SSCP analysis consists
of denaturating PCR products to avoid reannealing to its
complement; these fragments are then separated in non
denaturing polyaccrylamide gel electrophoresis (Orita et
al., 1989). The mobility of each strand is affected by its size
and its conformation. Therefore, the normal sequence DNA
of the wild type and the mutant DNA sequence appear with
different patterns. Recently, SSCP technology has been
widely used in both the detection and scanning of the
internal transcribed spacers (ITSs) for the studies of the
genetics of plant pathogenic microorganisms. Hence, the
ITS variation was used for differentiation more than 30
species of Phytophtora (Kong et al., 2003;2004) and more
than 40 species of Phythium (Kong et al., 2004). On the
other hand, this technique was used to differentiate
Rhizoctonia solani among others Rhizoctonia (Carling et
al., 2002) and Gaeumannomyces graminis (Goodwin et al.,
1995).
Random Amplified Length Polymorphism
(RFLP) and (PCR-RFLPs)
The random amplified length polymorphism (RFLPs)
belongs to the widely used hybridization developed in
Humans projects in 1980s (Botstein et al., 1980), and
thereafter in plant pathology research (Leung et al., 1993).
This technique reveals the pattern differences in the
sequences of nucleotides among different individual
organism. Moreover, these genetic differences are due to
evolutionary processes such as point mutation within the
fragment sequence, or through insertion, deletions or
inversion of sufficient size which could be detected by the
variation in the length of restriction fragments. The RFLP
analysis in its principles consists of DNA digestion with
restriction enzymes.
The resulting fragments are resolved according to the
molecular size through an agarose gel electrophoresis and
thereafter transferred to a nitrocellulose membrane by
southern blotting and visualized hybridization to a specific
probe (Southern, 1975). These techniques are highly robust
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with readily transferability between laboratories. On the
other hand, the PCR-RFLP technique consist of a
procedure that combine both the Polymerase Chain
Reaction(PCR) with anlysis of the restriction of fragment
length polymorphism (RFLP). Then, to detect
polymorphism within the ITS regions, an amplification of
restricted fragments was obtained using specific primers
(Fajardo et al., 2009).
Pyresequencing technology
The Pyresequencing technology is a DNA sequencing
by synthesis method in which a cascade of enzymatic
reaction whereby an enzymatic system are used (DNA
polymerase, ATP sulfurase, luciferase and apyrase) to
monitor the inorganic phosphate (PPi) that is released
during nucleotide incorporation on DNA chain extension
detected by luminometric system (Ronaghi et al., 1998;
Ahmadian et al., 2000; Ronaghi et al., 2000). This technique
takes advantages of the natural release of inorganic
pyrophosphate (PPi) whenever an nucleotide base is
introduced on the template strand. The released
pyrophosphate is then rapidly used by ATP sulfurylase to
convert APS (AminoPhosphonate) to ATP, which provides
the energy to luciferase to oxidize luceferin and produce
light, which is collected by a charge coupled device (CCD)
camera. The light produced is recorded in a peak program
known as pyrogram. The intensity and light of the peaks is
proportional to the number of dNTP incorporated. The
nucleotides that fail to incorporate are degraded by
apyrase between each cycle (Ahmadian et al., 2000). Mello
et al., (2011) has recently study pyrosequencing of the
ribosomal internal transcribed spacer-1 (ITS-1) with which
he has validated the effectiveness of such technology in
the survey of soil fungal diversity.
MALDI-TOF MS Technology
The high throughput genotyping method involving the
matrix assisted laser desorption/ ionization time of flight
(MALDI-TOF) is the amongst the more promising
technologies for generating SNP products Because of its
speed, cost effectiveness, and multiplex possibilities, The
MALDI-TOF procedure involves mixing the allele specific
products with the matrix on the metal surface (Nordhoff, et
al., 1996; Griffin et al., 1999; Griffin et al., 2000; Gut, 2001).
The analyte mixed with a saturated matrix solution forms
crystals. The matrix analyte are desorbed into a gas phase
where ionization is achieved by collision and accelerated
towards the detector and then separated by the time of
flight of each product. Ions with smaller mass-to-charge
ratios travel at a higher velocity than ions with a larger
mass-to-charge ratio (Griffin et al., 1999; Griffin et al.,
2000; Gut, 2001). MALDI systems are employed namely in
individual biotyping of numerous fungi species such as
Aspergillus, Fusarium, Penicillium or Trichoderma and
also various yeasts (Chalupov et al., 2013).
Setti and Bencheikh
In Silico ITS Mining
The large amount and the continuous accumulation of
plant pathogenic partial or total genomic sequences that
are now available in private and public databases could be
proposed as an alternative approach for both the de novo
ITS identification and also for ITS polymorphisms markers
studies (Bokulich and Mills ,2012 ; Porter et al., 2012;
Schoch et al., 2012; Toju et al., 2012). Thus with the
increasing of the freely databases, number of
bioinformatics pipelines including softwares, perl scripts,
packages modules are developed for interrogating,
detecting, retrieving and then list all ITSs automatically
(Bokulich and Mills, 2012; Schoch et al., 2012). This great
popularity of the computational method has led to the
construction of huge numbers of ITS databases for both
prokaryotes and eukaryotes organisms including
phytopathogenic microorganisms (Table1) (Birren et al.,
2002; DeSantis et al., 2006; Tripathy et al., 2006 ; Hedeler
et al., 2007; Kim et al., 2012; Quast et al., 2013; Cole et al.,
2014). These programs can be run on standalone computer
or as online version running on the web. Currently, more
and more pipelines are available in publicly domain. So,
we describe in the next section the most ones encountered
in the literature such CloVR-ITS (White et al., 2013);
FHiTINGS (Dannemiller et al., 2014); PIPITS (Gweon et al.,
2015); SCATA (Lindahl, 2011); PlutoF (Abarenkov et al.,
2010) and ITScan (Ferro et al., 2014 ).
CloVR-ITS (http://clovr.org)
The pipeline provides fungal microbiota for more
comprehensive studies for ITS amplicon sequence
analysis for fungal diversity studies. The CloVR-ITS
pipeline employs several well-known phylogenetic tools for
the analysis of ribosomal ITS sequence.To run the full
CloVR-ITS analysis track, at least two different input files
have to be provided by the user: a sequence file in fasta
format and tab-delimited metadata mapping file. Sequence
data may consist of a single fasta file or multiple fasta files
with trimmed and binned. Problematic sequences are
removed and consistency checks for correct formatting,
then quality filtering sequences are performed before
subsequent processing (White et al., 2013).
Fhitings(http://www.emerencia.org/fungalitspipeline.
html)
Is designed for use in rapidly identifying, classifying,
and parsing internal transcribed spacer (ITS) DNA. This
software is useful for fungal studies using next generation
sequencing (NGS). FHiTINGS is currently available on
UNIX-based platforms. FHiTINGS is an open source data
processing tool written in Python programing language
that rapidly facilitates the identification and taxonomic
classification of fungal ITS sequences. The software has
the ability to select, taxonomically classify, and quantify
the sequence abundance of database search results. Such
a pipeline exist for trimming and preparing sequences, and
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making multiple BLASTn-based database comparisons
with archived fungal specific databases (Dannemiller et
al., 2014).
Pipits
PIPITS is an open-source package available at
https://sourceforge.net/projects/pipits. A user-friendly
computational tool. The software is implemented in python
language. It is available as source code for Unix/Linux
environments. The stand-alone suite of software are an
ideal tools for both extraction of desired ITS subregions
from raw sequences and also exploits the RDP Classifier
for taxonomic assignment. PIPITS is the first automated
bioinformatics pipeline dedicated for fungal ITS sequences
which exploits the latest RDP Classifier to classify
sequences. Input files are generally provided as
demultiplexed FASTQ files where the Illumina machine
software splits the reads into separate files.The resulting
files are converted into a FASTA format(Gweon et al.,
2015).
Scata (http://scata.mykopat.slu.se)
SCATA is an efficient bioinformatic pipeline for
species identification and quantification after high-
throughput sequencing of tagged amplicons. It provids an
analysis framework for the analysis of sequenced tagged
amplicons, typically derived from high throughput
sequencing of microbial communities. scata system
support reading files from both IonTorrent and MiSeq
systems. Moreover, the Scata sytem can supports
uploading of sanger fastq files,. The fastq files can be
gzipped, which decreases the upload time. Merging of
paired MiSeq reads. Scata can now merge paired MiSeq
reads by aligning the reads and checking for overlap
(Lindahl, 2011).
PlutoF (http://plutof.ut.ee)
Is a cloud computing services for molecular
taxonomical and ecological studies. It also includes several
public web sites for displaying and searching the data.
these software packages are used for identification and
analysis of environmental ITS sequences of fungi, but it
can also operate on any other genetic marker and group of
organisms. The PlutoF web interface uses a series of
programming languages including PHP, HTML, CSS,
JavaScript, and SQL. Software currently available includes
massBLASTer, Fungal ITS Extractor, Chimera Checker and
454 pipeline for pyrosequencing data. (Abarenkov et al.,
2010).
ITScan (http://evol.rc.unesp.br:8083/itscan)
ITScan is an online and user-friendly automated
pipeline for fungal diversity analysis and identification
based on ITS sequences. It speeds up a process which
would otherwise be repetitive and time-consuming for
users. ITScan is an online and user-friendly automated
Setti and Bencheikh Transylvanian Review: Vol XXIV, No. 8, Special Issue, 2016

pipeline for fungal diversity analysis and identification
based on ITS sequences. It speeds up a process which
would otherwise be repetitive and time-consuming for
users. ITScan requires a FASTA-formatted input file
containing pre-processed sequences, i.e., high quality
sequences (usually Phred 20) without primer and
adaptor sequences. ITScan program was implemented in
Java and Perl with a user interface constructed using
Toolkit components. This software is platform
independent; it aruns on many systems including
Windows, UNIX and Linux. This pipeline uses others
modules such as BLAST and Phred(Ferro et al., 2014).

Table 1 : Some commonly plant pathogen genomics platforms and ITS databases with their websites.
Plant pathogen Host Web link References
database
EzTaxon database EzBioCloud http://eztaxon-e.ezbiocloud.net Kim et al., 2012
Ribosomal Database Michigan State University. http://rdp.cme.msu.edu Cole et al., 2014
Project
SILVA database Wolfgang Ludwig at the Technical http://www.arb-silva.de Quast et al., 2013
University, Munich, Germany,
Greengenes Center for Environmental http://greengenes.lbl.gov DeSantis et al.,
database Biotechnology 2006
Lawrence Berkeley National
Laboratory
Berkeley,
USA
Microbial database Virginia Bioinformatics Institute http://www.vbi.vt.edu/resources_and_t Tripathy et al.,
(VMD) ools/ 2006
Fungal Genome Broad Institute http://www.broadinstitute.org/scientifi Birren et al., 2002
Initiative (FGI) c-community/science/projects/fungal-
genome-initiative
e-Fungi Manchester University http://www.cs.man.ac.uk/~cornell/eFu Hedeler et al., 2007
ngi/database.html
MUMDB-MIPS Institute of Bioinformatics and http://mips.helmholtz-
DataBase Systems Biology muenchen.de/genre/proj/MUHDB/ Mewes et al., 2005
Helmholtz Zentrum Mnchen
Comparative Fungal Fungal Bioinformatics Laboratory, http://cfgp.snu.ac.kr Jongsun et al., 2008
Genomics Platform Seoul National University, Korea

Application of ITSs in Population Genetics
Studies
The ITS region undergoes unequal crossing over as
well as frequent insertion or deletion of the sequence
arrays and replication slippage makes them highly
variable and hence more informative (Baldwin et al., 1995 ;
Li, 1997). Moreover, the highly copy number facilitate the
amplificaiton and sequencing of this region (Baldwin et al.,
1995). The internal transcribed spacer (ITS), due to its fast
evolution has been used as reliable molecular marker in
numerous systematic studies including evaluation of
phylogenetic relationships among different taxa and is
being exploited for assessment of the genetic diversity.
(Baldwin, 1995; Peterson and Seberg, 1995; Bridge et al.,
2004). Moreover, ITSs markers have proved to be useful in
studies such as population structure and differentiation,
isolate and strain identification, fingerprinting and genetic
mapping (Gardes and Burns, 1993 ; Bridge et al, 2004;
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Choo et al., 2009; Peterson and Seberg, 1995). So, we
describe in the next section the summary of some major
application used in molecular plant pathology.
Species Identification and Strain Typing
Traditional methods for the detection, identification of
plant pathogenic microorganisms based on morphological
using microscopic, cultural and pathogenic
characterization constitute a key step with however
numerous limitations. Moreover, among filamentous fungi,
numerous species and isolates remain sterile on many
culture media. This will complicate more the identification
procedure due to the absence of reproductive strucutres.
On the other hand, closely morphological similar fungi are
often grouped under single specie. The reason why, an
alternative stable markers are necessary for the accurate
species identification which is essential for effective plant
disease management. Currently, the genetic markers are
Setti and Bencheikh
becoming increasingly used in plant pathology for
identification of individual at specific and intra-specific
levels (McLaughlin et al., 1981; Williams et al., 2001; Bridge
et al., 2003; Kristensen et al., 2006). It is presumed that
many plant pathogenic races, formae specialis or
pathovars differ from each other by only a few differences
of bases at different genes. The internal transcribed
spacer (ITS) region has been considered as a new
approach to differentiate fungi at species level. Moreover,
this region succefully generate specific primers that are
able to differentiate closely related fungal species (Bryan
et al., 1995). On the other hand, their high variability make
them a valuable tool for genus and species identification
(Gardes and Burns, 1993; Bridges et al, 2003).
Schneider et al., (1997), have already developed a
method for detection of Rhizoctonia solani isolates,
pathogenic and non pathogenic to tulips using the ITS
sequences. On the other hand, Henry et al., (2000), have
identified the fungus Aspergillus at species level and
differentiated it from others pathogenic and opportunistics
molds using ITS1 and ITS2 sequence regions. This could
have great impact on both the early diagnosis step but also
on the determination of the antifungal strategies. Ristaino
et al., (1998), have developped a PCR procedure to amplify
the ITSI and ITS2 sequence regions for quick identification
of the economically important species from each of the six
taxonomic groups in the plant pathogen genus
Phytophtora. Among the major work dealing the fungus
species identiification, is that deals with the Fusarium
species. In fact, Fusarium species are well-known plant
pathogens with significant economic impact. Fusarium is a
large, taxonomically complex genus. Since the majority of
fusarial isolates cannot be identified to species level by
traditional morphological methods, ITS molecular tools are
increasingly used to enable accurate species
determination (Wang et al., 2011). Furthermore, the ITS
sequences data has also given promising results for
species determination in majour powdery mildew
pathogens (Braun and Takamaton, 2000).
Assessment of Population Genetic Diversity
Studies
Studies of the genetic diversity of plant pathogen
population are the most significant development in
molecular plant pathology. These analyses are the most
important area in which a lot of effort has been made.
Furthermore, the polymorphic diversity could provide
significant information relating to the pathosystem.
Furthermore, the comprehension of the structure of plant
pathogen populations is important for understanding the
dynamic, the spatial distribution of the fungus to predict
and hence establish adequate control measures
(McDonald and Linde, 2002). On the other hand, the
comprehension of the structure of plant pathogen
populations is important for understanding the dynamic,
the spatial distribution of the fungus to predict and hence
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establish adequate control measures (Mc Donald and
Linde, 2002). The amount and distribution of genetic
variation within and among populations constitutes the
first step in studies of fungal population genetics.
Moreover, a high variation in fungal population can be
responsible for marked changes in pathogenicity and give
an indication of the level of fitness of the pathogen and
consequently its adaptability to the environment. These
variations are considered to pose a great risk for
overcoming control measures such as fungicide
applications and cultivar resistance (McDonald and Linde,
2002). In this context, Freeman et al., (1996) have examined
the population diversity using ITS sequence. This marker
was very helpful in determing the diversitiy in both inter
and intraspecific leve were isolates of Colletotrichum
gloeosporioides from tropical fruits, have revealed
considerable variation among isolates from avocado and
papaya. On the other hand, Gardes et al., (1991) and Bruns
et al., (1991) using the internal transcribed sequence (ITS)
regions working on ectomycorrhizal fungi discrimination
at species have succeed to separate the 26 isolates into 8
genera.
Recently, Oliveira and Cunha (2015) have studied the
structure of Erysiphe necator samples collected from
different regions of Portugal, where genetic analysis using
the amplification and sequencing the ITS spacers have
demonstrated that the populations of E. necator are
structured into two genetically distinct groups (A and B).
Assessment of Phylogenetics and Taxonomy
Studies
Due to the faster rate of evolution of the ITS region
than the coding sequences which vary among species
within genus or populations within species. This
characteristic makes the ITS markers an ideal candidate
for phylogenetic studies at various taxonomic levels
(Gonzalez et al., 1990; Sang et al., 1994; Baldwin et al.,
1995). Moreover, ITS markers have also been used to infer
the phylogenetic relationships up to closely interspecific
and intraspecific level based on measures of genetic
distances. Several studies have demonstrated that the ITS
marker are a valuable tool for phylogenetic analysis.
Already, Schnabel et al., (1999), using the ITS sequences,
has characterized Venturia inequalis and its relationship
with other tree fruit Venturia species.
Furthermore, using both the ITS1 and ITS2 rDNA, the
authors fonund that this molecular marker have divided
the Venturia species in three monophylitic groups linking
to their corrresponding host rather than to thier
anamorph. Others studies concerning the relationship
between strains of the Venturia species have revealed the
that the Venturia species are placed in three distinct
monophylitic groups in a phylogenetic tree.namely the
V.inequalis, V. Pyrina and the third V.cerasi (Shnabel et al.,
1999). On the other hand, different isolates of the genus
Verticillium, representing 13 species of diverse
Setti and Bencheikh
econutritional groups were examined using the ITS regions
to construct phylogeny tree. The authors suggested that all
the plant pathogens (V. alboatrum, V. Dahlia and V.
Nigrescens belong to the same clade (Bidochka et al.,
1999). Ward et al., (1994), using the ITS region, have
resolved the taxonomic relationship between Polymexa
species namely P. betae and P.graminis. the study
confirmed that both species are phylogenetically distinct.
Moreover, the P.graminis could be divided into two
subgroups.
Concluding Remarks
Advances in the development of molecular tools
especially those using PCR technology have provided a
pannel of methods for wide array of application for fungus
population studies including reliable identification,
phylogenetic relationships and taxonomy and genetic
diversity. Because of their abundance, reproducibility,
inheritance and the rapidity of their detection, the ITS
markers are the major tool indicated for the genetic
population studies. Meanwhile, the major difficulty with
the ITS is their discovery and their detection which is still
more expensive and labor intensive. However, with
increasing of private and public databases, a new
alternative method based on bioinformatics approach has
become increasingly used for many plant pathogenic fungi.
Different bioinformatic softwares, are now available for
interrogating, detecting and retrieving ITSs. All these
previous cited features make of ITSs an ideal candidate
DNA region that can be used in solving practical problems
including genetic diversity, population structure gene
tagging, phylogenetic and taxonomy. On the other hand,
with increasing quantity of sequenced organisms, ITSs
region will be expected to have a key role in genomic
research and particularly in population genetics studies.
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