American Journal of Plant Sciences, 2013, 4, 1375-1386 Published Online July 2013 (
Molecular Genetic Diversity in Iranian Populations of
Puccinia triticina, the Causal Agent of Wheat Leaf Rust
Seyed Taha Dadrezaie1,2, Samer Lababidi3, Kumarse Nazari3, Ebrahim Mohammadi Goltapeh1,
Farzad Afshari2, Fida Alo3, Masoud Shams-Bakhsh1, Naser Safaie1
1College of Agriculture, Tarbiat Modres University, Tehran, Iran; 2Seed and Plant Improvement Institute, Karaj, Iran; 3International
Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria.
Received April 16th, 2013; revised May 17th, 2013; accepted June 10th, 2013
Copyright © 2013 Syed Taha Dadrezaie et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Wheat leaf rust caused by Puccinia triticina, is the most common and widely distributed wheat rust in the world. In
order to study the genetic structure of leaf rust population 14 pairs of AFLP and 6 pairs of FAFLP primers evaluated on
86 isolates samples collected in Iran during spring of 2009. Results showed that almost all investigated isolates were
genetically different and special pattern of AFLP allele’s that confirm high genetic diversity within leaf rust population
was observed. Analyses showed, all provinces were classified into three major groups particularly similar clusters were
found between then neighboring provinces. Rust spore can follow the migration pattern in short and long distances to
neighbor in provinces. Results indicated that the greatest variability was revealed by 97% of genetic differentiation
within leaf rust populations and the lesser variation of 3% was observed between the rust populations. These results
suggested that each population was not completely identical and high gene flow has occurred among the leaf rust popu-
lation of different provinces. The highest differentiation and genetic distance among the Iranian leaf rust populations
was detected between leaf rust population in Sistan and Baluchistan and highest similarity was observed between in
Ardabil provinces. The high pathogenic variability of leaf rust races in Ardabil and Northern Khorasan may be an indi-
cation that these two regions are the center of origin of pathogenic arability. Present study shows that leaf rust popula-
tion in Iran is highly dynamic and variable.
Keywords: Leaf Rust; Gene Resistance; Genetic Diversity; Puccinia triticina; AFLP and FAFLP Markers
1. Introduction
Wheat leaf rust caused by Puccinia triticina Eriks (Pt), is
the most common and widely distributed wheat rust in
the world. Leaf rust damage is less than yellow and stem
rust, but level of brown rust damage causes greater an-
nual losses due to its more frequent and widespread oc-
currence [1]. Despite extensive studies and scientific re-
markable success, in reduingce damage in leaf rust in the
twentieth century due to changes races still heavy losses
to this valuable product is being imported. Damage the
disease has been reported from different parts of the
world. In 2007, leaf rust in Kansans’ winter wheat was
decreased by 14% product, which affected production. In
susceptible cultivars loss yield was estimated at more
than 50% [2]. Wheat leaf rust is present in all wheat
growing areas in Iran and a large number of races were
found in a recent study of pathogenic variability of Pt
population in Iran [3,4].
There are so many ways to control the disease, of the
genetic resistance a preferred method for reducing dam-
age leaf rust, evaluation of genetic diversity and popula-
tion structure within species of fungi plant pathology in
order to achieve resistance and its management is very
essential. Therefore, understanding of the genetic struc-
ture of pathogens and identification of pathogenic viru-
lent factors in disease is primarily important. On the
other hand ability to produce new races, proliferation and
their ability to reproduce rapidly and the epidemic of the
important features of the rust, compared with other dis-
eases that cause them to be more important and it is nec-
essary to constantly change pathogen races.
Characteristics of virulent on resistance genes is more
important application of, races analysis in the world is
still more influential, Because these virulent properties
are under strong selection. It is likely that these pheno-
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Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
typic characteristics provide incorrect estimate of the
potential genetic variation of pathogen populations. Va-
riety of molecular markers based on the DNA that in
plant pathogens are used for the study population. Using
AFLP markers for segregation in the P. triticina has been
conducted in different countries. On the other hand ure-
dospores migration to new areas occurred by wind and
air currents. In fact the important feature is that rust are
travel over the continent and uredospores can spread by
wind hundreds kilometers away from the infected plants
source. Those factors are important for genetic diversity
and occurrence of new virulence to the region. Therfore
the investigate relationship pathogen races of in prov-
inces with air flow path was the main objective of this
A study on American virulence phenotype and ampli-
fied fragment length polymorphism (AFLP) markers
were used to examine 42 isolates collected between 1960
and 2004. The data indicated that the contemporary iso-
lates (collected since 2000) were very distinct from older
isolates (collected before 2000) based on virulence and
AFLP markers, and that the old population prevalent
before 2000 may have been replaced by the contempo-
rary population. The old and new populations appear to
be genetically distinct and may represent an exotic in-
troduction rather than a mutation in isolates of the old
population [5]. Kolmer et al., [6] tested 64 isolates for
randomly amplified polymorphic DNA (RAPD). Fifteen
different RAPD phenotypes of the fungus were distin-
guished using 10 polymorphic DNA bands among 64
isolates. However, there were 37 virulence phenotypes of
the fungus as determined by 19 near-isogonics differen-
tial lines of wheat. RAPD markers have been useful for
characterizing variation between regional groups of P.
triticina virulence phenotypes but less so for detecting
variation between closely related virulence phenotypes
within or between regions [7]. A comparison, to study
the population genetics of yellow rust in Gansu and
Yunnan two China Provinces, with AFLP method was
applied. Forty one AFLP genotypes were obtained from
150 isolates. Genotypic diversity in Gansu Province was
higher than that in Yunnan Province. A free recombina-
tion signature was detected in Gansu Province but not in
Yunnan Province [8].
Effective breeding for disease resistance requires ex-
tensive information on the incidence and virulence of
endemic pathogens; which is of high importance for rust
fungi as they are able to rapidly evolve new virulent
races. Knowledge on the diversity of the leaf rust fungus
in Iran is limited; and, information on the diversity of P.
triticina isolates regularly occurring in Iran is also rare;
to study the molecular diversity of Iran P. triticina iso-
lates, amplified fragment length polymorphism (AFLP)
markers have been successfully used in mycology and
plant pathology for the differentiation of species within
genera [9-11]. Because these markers are not neutral and
under selection in contrast to resistance-specific viru-
lence, which is subject to strong host selection are as
more accurate tools for genetic structure study [12-14].
The FAFLP (Fluorescent AFLP) method, a large number
of cut pieces of genome replication was labeled then
these fragments were separate and distinct parts in a ma-
chine sequencer [15]. AFLP and FAFLP markers were
used for this study to investigate the genetic variability of
leaf rust isolates collected from 14 province of Iran. Our
general hypothesis was that genetic similarity within
populations of 14 provinces would be considerably
higher than between populations. This hypothesis might
have been nullified by the existence of isolates of P.
triticina. Our objective was, however, to study the gen-
eral molecular diversity of P. triticina isolates collected
from different part of Iran. No study has been done on
molecular diversity in leaf rust in Iran before. This study
to assess the genetic diversity between and within leaf
rust populations in various provinces of the country using
the AFLP and FAFLP molecular markers was performed
in 2010 at ICARDA. These markers can be easily
searched in whole genomes and this was attempts to ge-
netically related fungi based clear geographical distribu-
tion of air flows.
2. Materials and Methods
2.1. Leaf Samples Collection, Purified and
Amplified Samples Collected
About 100 samples of leaf rust-infected leaves were ran-
domly collected from 14 provinces of Iran in early spring
of 2009. The samples were dried in open air and then
were kept in a refrigerator at 4˚C. The isolate were
gradually regenerated on the susceptible Bolany cultivar
and the spores were amplified. The initial proliferation
was considered as a spores bulk or mass of the popula-
tion. From each spores masses 5 to 7 pots were inocu-
lated and from each pots 3 to 5 single pustule were ran-
domly selected and amplified. To ensure purity all single
pustules were single pustulated 2 or 3 times. 8 to 10 old
seedlings of susceptible Bolany cultivar were used for
2.2. Determination of Virulence Phenotypes
Pathotype identification based on the formula Virulence/
Avirulence 38 differentials lines of Thatcher in seedling
stage were tested. Thatcher wheat lines with single resis-
tance gene were used as follows: Lr22b, Lr1, Lr2a, Lr2b,
Lr2c, Lr3, Lr3ka, Lr3bg, Lr9, Lr10, Lr11, Lr12, Lr13, Lr14a, Lr14b,
Lr15, Lr16, Lr17, Lr18, Lr19, Lr20, Lr21, Lr22a, Lr23, Lr24, Lr25,
Lr26, Lr10, 27+31, Lr28, Lr29, Lr 30, Lr32, Lr 33, Lr34, Lr35, Lr36,
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Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust 1377
Lr37, Lrb all isolates were evaluated on all the above
genes and also physiological races of leaf rust were de-
termined based on modified uniform nomenclature sys-
tem. Using the results of above 20 lines which mostly
isogenic in 5 tetramerous groups based on Ordonez et al.
2010 [2]. Bolany was as a susceptible check. To evaluate
the seedling stage in inoculated plants, types of consid-
ered infection were created on the lines and recorded 12
to 24 days after inoculation according to McIntosh et al
(1995) method [16]. Infection types 0 to 2+ were classi-
fied as avirulent and infection types 3 to 4 were classified
as virulent. Pathogenesis frequency for resistance gene
was calculated.
2.3. Determination of Molecular Phenotypes
2.3.1. D NA Extracti on
DNA extracted from fresh urediniospores by using a
modified CTAB procedure (Hexadecyl trimethyl ammo-
nium bromide) as follows steps: 1) Weight 50 mg spores,
50 mg small ball and two small balls (derided spore in
freeze drier for 6 - 8 days). 2) Place tubes into liquid ni-
trogen for at least 30 seconds before continuing to step
three. 3) Put the tube in amalgamator and tape tightly
into place turn on for 40 seconds (30 shaking in each
seconds). 4) Repeat steps 2 and 3 two time again (in to-
tally three time). 5) Add heat (65˚C) CTAB 2% 1200 µl
and place them in the water bath (65˚C) for 30 - 45 min-
utes (during the incubation, mix each tube, one at a time,
by inverting perform tube mixing three times during the
incubation period and placed tubes in ice for 5 minutes. 6)
Add 600 µl chloroform/isoamyl (24:1) mix tubes by gen-
tle inversion for 2 minute (100 - 120 inversion). 7) Cen-
trifugation for 12 min at 13,000 rpm in 4 temperature
transfer aqueous phase to 2 ml eppendorf tubes. 8) Add
100 µl R40 and incubate at 37˚C for 60 minutes. 9) Add
600 µl chloroform/isoamyl (24:1) mix tubes by gentle
inversion for 2 minute (100 - 120 inversion). 10) Cen-
trifugation for 12 min at 13000 rpm in 4 temperature
transfer aqueous phase to 2ml eppendorf tubes. 11) Add
100 µl of 3M sodium acetate PH 4.8. 12) Add 600 µl
cold Isopropanol and then mix gently by hands keep it in
freezer 20˚C for min. 13) Centrifugation for 12 min at
13,000 rpm in 4 temperature transfer aqueous phase to 2
ml eppendorf tubes. 14) Wash with cold ethanol 75%
with 1000 µ. 15) Centrifugation for 12 min at 13,000 rpm
in 4 temperature transfer aqueous phase to 2 ml eppen-
dorf tubes. 16) Dry the pellet well on paper towel for 20
minutes. 17) Add 100 µl of TE buffer to resuspend and
keep in Freezer.
2.3.2. AFLP
For analysis of genetic diversity, AFLP markers were
applied on population samples based on Vos et al., 1995
[17]. Restrictive digestion enzyme and PCR method were
used in this method. AFLP protocol was followed as de-
scribed in CIMMYT applied molecular protocol [18].
2.3.3. Selectiv e PCR
For use in stage of selective amplification initial repli-
cating stage of the samples were diluted 5 times. In this
study, Initially based on the results of previous work re-
lated to the rust and in other cases more than 40 pairs
combination of leaf rust on four selective sample (Safi-
Abad Dezful, Mehran, Bayea kola and Ajab sheir) were
tested. Then created based on polymorphism 14 pairs
combination were selected and evaluated on all samples
were analyzed on. All samples were about 100, because
of limitations in the gel sample was reduced to 86 (Table
1) AFLP primers combinations used in this study: 1 =
P02 + M51, 2 = P10 + M183, 3 = P11 + M50, 4 = P32 +
M50, 5 = P11 + M47, 6 = P24 + M17, 7 = P24 + M54, 8
= P37 + M50, 9 = P16 + M47, 10 = P13 + M301, 11 =
P16 + M183, 12 = P16 + M183, 13 = P16 + M88, 14 =
P16 + M17.
2.4. Evaluation 6 Pairs of FAFLP Primer in ABI
Electrophoresis System
To complete study in order to accuracy of the previous
results, six pairs primers of AFLP markers labeled with
fluorescent dye (FAFLP) were used, then separated the
dye-labeled fragments by capillary electrophoresis, this
method is currently one of the most suitable methods for
fragment analysis in automated ABI Prism 3100/3100-
Avant sequencer (construction company, Applied Bio-
systems). Just one of the primer combinations fluores-
cent-labeled was used. Primers were labeled by three
kinds of fluorescent dye, FAM blue, Ned yellow and
green Vic in this study. During the reaction, fluorescent
dye is not involved in amplification process and it's only
used for peak observation (bands) in the ABI. For this
purpose, eight P primers (P10, P11, P14, P15, P16, P19,
P32, P40, and P41) which by various fluorescent were
labeled and with three M primers (M54, M55, M54),
make 24 combinations of primers. Based on experimen-
tal results, six primer pairs were selected, this primers
including (P11/M54; P11/M55; P10/M54; P10/M55;
P14/M54; P19/M55). PCR program for testing the la-
beled AFLP markers were used exactly similar to con-
ventional PCR in the AFLP that previously described.
After the primary amplification, of the three pairs of
samples of different combinations with different color
fluorescent mix (ABI electrophoresis system due to dif-
ferent fluorescent, three primers in each well will be de-
tected) was diluted in water. From this mixture diluted
and uniform, only one micro liter were taken and to new
platform are that each well contains 5 μl standard for-
mamide (Hi-Di formamide) was transferred and pipette.
Formamide standard (Hi-Di formamide) containing Rox
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Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
Copyright © 2013 SciRes. AJPS
Table 1. Characteristics of leaf rust isolates collected form differant parts of Iran in spring of 2009.
Isolates No. Location Race Isolates No. Location Race
1 Ahvaz MGHSS 44 Moghan (Ardabil) FKTRS
2 Ahvaz MGMSN 45 Moghan (Ardabil) BJKFJ
3 Ahvaz DBGTJ 46 Moghan (Ardabil) CJHCS
4 Susangerd MBMTJ 47 Moghan (Ardabil) PGRKS
5 Lali MBHNB 48 Karaj PJTRS
6 Safi-Abad (Dezful) MJHTS 49 Karaj PJTTS
7 Safi-Abad (Dezful) BBJNG 50 Karaj FDRRS
8 Safi-Abad (Dezful) CBBTJ 51 Karaj FFTTS
9 Mehran CGTRS 52 Karaj FJQRS
10 Mehran BBGSG 53 Boroujerd FJRSS
11 Mehran DBGTJ 54 Boroujerd PKRTS
12 Mehran DBGTJ 55 Boroujerd FKRRS
13 Safi-Abad (Dezful) FHQSS 56 Boroujerd FFTTS
14 Baykla (Sari) FKTMS 57 Neishabour FKTRS
15 Sari MJTRS 58 Neishabour FKTRS
16 Qrakhyl (Sari) CJRCS 59 Zarghan FJRRS
17 Kelardasht FJRMS 60 Zarghan FKTTS
18 Mashhad FKTRS 61 Zarghan FKTRS
19 Qrakhyl (Sari) MHSTS 62 Mashhad FKTTS
20 Baykla (Sari) MKSTS 63 Ardabil FJRRS
21 Baykla (Sari) MKSKS 64 Ardabil FJTRS
22 Baykla (Sari) FKTHS 65 Ardabil PKTRS
23 Sari FHTTS 66 Gorgan CGRMQ
24 Gorgan
25 Gorgan MKTTS 68 Gorgan FGRRQ
26 Gorgan PKTTS 69 Ajabshir FGHMQ
27 Gorgan FKTSS 70 Bonab FGHLQ
28 Islamabad FKRKS 71 Kelardasht DHRTS
29 Islamabad FKRKS 72 North Khorasan FGRRQ
30 Moghan (Ardabil) MDSKS 73 North Khorasan FGRRQ
31 Moghan (Ardabil) MKSJS 74 North Khorasan PGTRS
32 Moghan (Ardabil) MKKTS 75 North Khorasan FGTMQ
33 Moghan (Ardabil) MJTHS 76 North Khorasan FGRMQ
34 Moghan (Ardabil) FKTRS 77 North Khorasan FGRMQ
35 Moghan (Ardabil) FKTRS 78 North Khorasan PJTTS
36 Eshtehard CHRMN 79 North Khorasan PJRTS
37 Marivan FKTKS 80 Moghan (Ardabil) FHRRS
38 Marivan FKRHS 81 Moghan (Ardabil) FHTRS
39 Marivan CHQKS 82 Zabul BJRRS
40 Marivan FKRHS 83 Zabul FJRRQ
41 Islamabad FKRMS 84 Ahvaz CJTQQ
42 Moghan (Ardabil) CJRRS 85 Anchusa (Moghan) FJRRS
43 Moghan (Ardabil) MFTTS 86 Barley leaf rust PHRRQ
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust 1379
which has no color but red fluorescence and in the sys-
tem ABI electrophoresis is to produce a red band. Stan-
dard formamide is actually contained ladder in the stan-
dard formamide itself is Gs 500 Rox size standard and
are used as indicators to measure. Standard size 500 is
designed to measure the DNA fragments. Gently pellet
material was mixed well and PCR 96 platform with the
specific PCR (Mat) were covered. In order to denaturing
4 minutes at 95˚C in PCR machine was placed. Plate
immediately transferred into ice and was kept on ice until
placed in the ABI.
2.5. Data Analysis
2.5.1. Stati s ti cal Analysis of Data Obtained from
Ordinary AFLP
The presence or absences of bands in the range 100 to
800 bp, respectively, were allocated to one and zero.
Only bands with high resolution were considered. Si-
multaneously assessment of phenotypic and genotypic
parameters, Correlation analysis of factor analysis and
cluster analysis was performed.
Cluster analyses were done Jaccard’s similarity coeffi-
cient and UPGMA algorithm, UPGMA and Clustering
options with the software NTSYS-PC ver. 2.02. and Dar-
win was performed Bootstrap validation tree branches
with 1000 replicates was determined.
2.5.2. View and Analyze the Results of the
Electrophoresis System ABI
Automated sequencer system based on capillary electro-
phoresis works sensors using capillary, samples were
prepared and evaluated. Results are analyzed automati-
cally moved to a computer connected to the machine.
Information output machine using the 3.5 software
genotyper electropherogram was shown.
Electrophoresis machine for analysis of data from ABI,
results of electrophoresis on a computer connected to the
machine to another computer that was taken with Gene
Mapper software transferred. Electrophoregram output
sequencer machine using Gene Mapper software were
analyzed. In this electrophoregram horizontal and verti-
cal scale determines the size of respectively piece of
DNA (bp) and relative fluorescence signal intensity or
concentration requirements.
In Gene Mapper software data with definitions such as
ladder type markers, dye size and profile obtained from
peak PCR amplification of samples obtained from each
well was done. The binary data matrix was performed
(One as present DNA band or Peak and zero means ab-
sence of band or peak). Analyses of molecular variance
AMOVA were performed with software Power Marker
and calculate the variance components and each of pro-
portion in the total variation was determined. Then data
in order to grouping Materials into cluster analysis based
on Jaccard similarity coefficients and UPGMA method
was performed by software NTSYS2.1.
3. Results
The results of the genetic relationship between popula-
tions of leaf rust of wheat in Iran
3.1. Molecular Evaluation
Analysis of Band Patterns: In review and analyze the
band patterns only a couple clear and complete resolution
bands were scored. Results from the proliferation of
bands Amplified bands from 14 primer combinations on
polyacrylamide gel showed that 476 clear bands were
produced in total, which the 129 polymorphic bands (Ta-
ble 2).
3.2. Cluster Analysis and Dendrogram Drawing
Data from 14 pair’s primers with Jaccard similarity coef-
ficient, cluster analysis was performed with software
NTsys (Ver. 2.02) and Darwin based on the UPGMA
algorithm in the end results as a dendrogram were sum-
marized. Based on the results of dendrogram in both
software, 86 leaf rust isolates to 8 main groups with 30%
similarity level was divided. By calculating the correla-
tion coefficient cophentic determined the resulting den-
drogram revealed high similarity with the data matrix.
Table 2. The total number and polymorphic produced by
primer combinations.
Row Primer
The total number
of bands
1 P02 + M51 10 41
2 P10 +M183 8 25
3 P11 + M50 20 59
4 P32 + M50 3 28
5 P11 + M47 4 32
6 P24 + M17 11 40
7 P24 + M54 18 38
8 P37 + M50 12 20
9 P16 + M47 7 45
10 P13 + M301 11 20
11 P16 + M183 3 34
12 P38 + M47 5 30
13 P16 + M88 7 22
14 P16 + M17 10 42
Total 129 476
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Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
Also, separately data from six primer pairs labeled
with software NTsys (Ver. 2.02) and Darwin based on
UPGMA algorithm cluster analysis was performed. The
isolates were grouped with the Jaccard similarity coeffi-
cient and was determined and by calculating the correla-
tion coefficient cophentic that the data matrix has a high
similarity (r = 0.82) with this dendrogram. Based on re-
sults of the data labeled primers the isolates were divided
into nine groups.
Often groups in both cluster had overlapping together
but more importantly, the provinces that have been iden-
tified as important areas of brown rust such as Ardebil,
Khuzestan, Mazandaran, and Golestan North Khorasan
were present in most groups and Indicates that diversity
was found within the province. Both data merged to-
gether and again perform cluster analysis, and its Den-
drogram was drawn. Eleven groups were formed. Those
eleven separate groups were formed in 28% similarity
and indicate a large diversity in groups. The results based
on molecular data showed that diversity of leaf rust iso-
lates in Iran is very high. The first groups were included
33 isolates where from all the provinces apart of Sistan-
Baluchistan province, there were one or more isolates.
The second group was consisted of five isolates from the
four provinces of Fars, Sistan and Baluchistan, Khuzestan
and Khorasan. The third group included one isolate from
the Lorestan and the eleventh group was Moghan isolates
from Anchusa. italica in a single group was formed a
sister group with other groups again.
AFLP data on polyacrylamide gel and sequencer also
showed very high diversity. For a more complete deter-
mined genetic relationship between isolates and similar-
ity and better closeness between them and also investi-
gate probability of molecular markers associated with
virulent isolates, virulence and virulence data of 86 iso-
lates of leaf rust on 38 genes resistance to leaf rust was
analyzed with software NTsys (Ver. 2.02) and Darwin.
This analysis was based on UPGMA algorithm but iso-
lates with the simple similarity coefficient were grouped.
Dendrogram obtained by calculating the correlation co-
efficient cophentic was found to the data matrix has a
high similarity (r = 0.82). Simple coefficient is used in
phenotypic characters (Figure 1).
Based on the results of the data virulence and a viru-
lence isolates were in thirteen groups. The first group
included 16 isolates; the second group included 5 iso-
Direct and individually correlation from virulent data
groups with groups derived from molecular data was not
obtained, but all data and analysis indicate that diversity
is very high in the samples investigated and confirmed
the correlation between neighboring provinces.
More than 65% of isolates were virulent on 17 to 23
genes. One isolate was virulent on 26 genes and one iso-
late was also virulent just on six genes. This isolates ei-
ther in terms of the on which genes resistance are viru-
lent and both can be pathogenic on number of resistance
genes have been varied. Regarding that which terms of
the genes that are pathogenic and on which genes not
virulent in 64 races were placed and regarding can be
pathogenic to a number resistance genes were classified
in 19 groups (Figure 2).
In this results are not a significant relationship be-
tween populations and geographical distribution that one
particular group is belongs to one particular region or a
province. But there were several different groups in
provinces and in large groups are present all provinces,
which suggests that the in provinces population diversity
is high and from this views are the same population. In
fact the provinces were grouped together. This was most
closely related to neighboring provinces (Table 3, Fig-
ure 3).
3.3. Diversity and Genetic Distance between
Based on data from the markers labeled in sequencer
Table 3. Number of ineffective genes and Number of iso-
Number of ineffective genes Number of isolates
26 1
25 1
24 3
23 9
22 9
21 8
20 11
19 5
18 7
17 7
16 5
15 4
14 2
13 5
12 3
11 2
10 1
7 2
6 1
Copyright © 2013 SciRes. AJPS
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
Copyright © 2013 SciRes. AJPS
Figure 1. Dendrogram derived from analysis of AFLP bands on polyacrylamide gel and ABI machine of 86 leaf rust isolates
using UPGMA method and Jaccard’s similarity coefficient.
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
Figure 2. Dendrogram based on data from 86 leaf rust isolates virulent on 38 gene leaf rust resistance.
1003 the polymorphic alleles are identified, analysis of
molecular variance and genetic distance on this data was
calculated by the software Power Marker (Table 4).
Results of Analysis Molecular of variance showed that
the genetic differences within populations are 97% and
difference between populations is 3% and very high gene
flows between populations exist. Genotypic diversity
among the populations studied was different so that the
Ardebil population with 81% and Fars population with
50% respectively had the highest and lowest genotypic
diversity. Maximum of the genetic distance between
populations of leaf rust isolates in Iran’s was belonging
to Sistan-Baluchistan province, After that Golestan and
Kermanshah provinces had the highest genetic distance
with the other (Figure 4). Leaf rust population of Ardebil
Isolates was more near with other populations of leaf rust
in Iran, which North Khorasan was with the lowest dis-
tance. After the North Khorasan Ardebil had the highest
similarity with other province. It seems these two areas
are centers of leaf rust diversity and distribution in Iran.
Copyright © 2013 SciRes. AJPS
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust 1383
Figure 3. Pathogenic potential of different isolates studied
on resistance genes.
Figure 4. Dendrogram obtained from cluster analysis of leaf
rust populations using Nei genetic distance and the UP-
GMA algorithm.
Table 4. The calculated values for genetic diversity betwee n
and within populations of leaf rust.
Source Df SS MS Est. Var. %
Among Pops 12 1105.98392.165 2.681 3%
Within Pops 73 5488.08775.179 75.179 97%
Total 85 6594.070 77.860 100%
4. Discussion
The results showed that almost all the studied isolates
were genetically different, all studied P. triticina isolates
were produced unique AFLP alleles patterns, which the
high genetic diversity within population of the leaf rust
fungus was described by McDonald and Linde 2002 con-
firmed [19]. Also this research confirmed results of
pathotypes and physiological races of P. triticina in Iran
conducted by Dadrezaei et al., 2012 that there was great
diversity in Iranian races of leaf rust. Their results
showed that among 234 single, 177 different virulence
classes (races) were identified [4].
In Canada Kolmer 69 isolates collected, and were
evaluated by 10 pair’s combination of AFLP. Only 37
distinct phenotypes virulent with Thatcher lines were
detected, whereas 69 Molecular phenotypes with assis-
tance 164 AFLP markers were detected [7]. In another
study in Morocco form four different farming-ecological
areas leaf rust isolates collected and were evaluated with
5 pairs AFLP primer combinations. Analysis of molecu-
lar variance of AFLP pattern showed no significant dif-
ferences among populations. But diversity within popu-
lation was very high. Patterns of genetic diversity among
isolates collected indicate high gene flow and a complex
system of reproduction. Leaf rust isolates in Morocco
have sexual and asexual production stages and sexual
stage is on Anchusa. italica [20].
Reproductive system and gene flow are important fac-
tors that determine their genetic structure. The wheat
rusts are heteroecious, and therefore requires a telial/
uredinial host (usually wheat) and an alternative (pycnial/
aecial) host (for leaf rust are) Thalictrum speciosissimum,
Isopyrum fumaroides or Anchusa italica to complete the
full life cycle. Anchusa and Thalictrum have been re-
ported in Iran, but no study or report on sexuality pro-
duction in Iran exists. Probably sexuality form leaf rust is
prevalent in Iran so probably part of the genetic diversity
observed in population of P. triticina may be due to re-
combination. This study showed that the contrary to
Torabi et al. 2001 [21] which declared that genetic diver-
sity of leaf rust in Iran was not very high, genetic diver-
sity leaf rust in Iranian isolates is very high and accord-
ing to research results in different world. In this study to
separation and analysis of the different races of leaf rust,
both characteristics of virulent on resistance genes and
molecular markers were used. Both results indicate and
confirmed very high variation in pathogenicity and ge-
netic diversity in pathogen. Characteristics virulent on
resistance genes the importance, more applicable, more
useful in races analyses and are still in use, But because
virulent properties are under strong selection it is likely
provide incorrect estimate the potential genetic variations
pathogen populations. But variety of DNA-based mo-
Copyright © 2013 SciRes. AJPS
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust
lecular markers have been used to study populations of
plant pathogens, because these markers that are selec-
tively neutral, highly informative more accurate tool and
it is best to use for studying the genetic structure and
because analyses are based on allele frequencies, it is
vital adequate to make reliable estimates [12].
Pattern of pathogenicity in the province showed that
there are the common races in different province of
countries exist. Also on the air streams flowing in spring
and at track races in this trace, probably has race connec-
tion between the southern regions, South West, West,
North West, North and North East of country. This
grouping is based on the relationship between patho-
genicity and non pathogenicity on 38 Thatcher isolines
that had been discussed previously was also confirmed in
these results. But the relationship more clearly and pre-
cisely by dendrogram was created from the molecular
markers. By exact examining dendrogram and its nu-
merous subgroups can be detected three adjacent prov-
inces that were most similar or near together could ob-
serve. In better words three similarities can be identified
in three domains South West to North West, North West
to North East and North-East to the South East, and that
most isolates in the adjacent provinces in the dendrogram
are located together. When carefully observed to these
pathways they are affected by air mass that Iran is fully
compatible correspond and way they are affected by the
Zagros and Alborz Mountains range. The main air
masses during the wheat growing season will affect Iran
include Mediterranean air masses that enter the country
from the West and North West. Iran is out of the East
and most parts of the country will affect its precipitation.
The flow direction before entering Iran will pass Leba-
non, Syria, Turkey, and Iraq. The second is related to the
Sudanese air mass that form Africa, Yemen and Saudi
Arabia and crossed over into the Persian Gulf will be
entering in Iran. These two air masses have very
important role to transferring urediniospores in wheat
growing areas. This direction is the same direction that in
the 1990s makes transferring of yellow rust spores had
virulence on gene Yr9. The Yr9 virulence detected in
Ethiopia in 1986 migrated to Yemen in 1991 then to
Egypt and West Asia such as Syria and Iran (1991-1992)
and reached Indian sub-continent (1996) over a period of
10 years riding on wind currents from west to east. This
caused severe crop losses in widely grown cultivars cov-
ering more than 20 million hectares. These air masses
will cause yellow rust epidemics in the CWANA region
and heavy damage to wheat. For example estimated grain
losses were in the order of 1.5 million tons in 1993 and
one million tons in 1995 [22]. Such new reaces introduc-
tions of leaf rust and yellow rust in this area is probable
Third air mass affected Iran is Siberian air mass. It is
entered from the north of Iran. This air mass past to the
countries of the Caucasus, Kazakhstan, Uzbekistan and
Turkmenistan across the Caspian Sea and causes the
highest rainfall in the north of the country. Existence of
the identical races especially in the adjacent provinces
along with the existence of air flows on this way rein-
forced the reason of linking of these provinces through
air flows. This relationship was both in grouping based
on being virulent or non-virulent on the seedling of
wheat leaf rust near isogenic lines and in the abundance
of the races. The aggressive isolates in rather similar
groups showed the relation between the provinces. Al-
most most of the provinces were related to each other.
This suggests the gene flow and vast and effective trans-
mission of spores through air flows [4]. There are nu-
merous examples of mutations and migration that cause
appearance and rise of non-indigenous races form un-
known areas. Non-native races durum wheat in the last
decade in Europe and North America has been attacked.
In Rabbani Nasab studies genetic similarity of most
Iranian wheat yellow rust populations with West and
North West yellow rust populations (including the prov-
inces of Elam, Hamadan, Kermanshah and Ardebil) more
than 65 percent. He caused the occurrence of gene flow
due to genetic similarity population to each other’s [23].
Genetic diversity analysis of yellow rust isolates in
Denmark, England, Germany and France use to AFLP
showed that great Yellow rust spores immigration are
occurrence between Denmark, England, Germany and
France [24]. Phenotypic and genotypic results of differ-
ent studies, including traps nursery and various molecu-
lar markers studies showed that leaf rust spores can be
exchanged between the America and Mexico and dem-
onstrate transfer of spores moving from America to
Mexico and probably from Mexico or the Southwest Pa-
cific to America [1,2,25,26].
Analysis of molecular variance using the AFLP data
indicated that the greatest variability was revealed by
97% of genetic differentiation within leaf rust popula-
tions and the lesser variation of 3% was observed be-
tween the rust populations. Although the high molecular
variability within the rust population may indicate the
colonial behavior of leaf rust in distinct provinces, but
similarity between rust populations indicates the gene
flow. These results suggested that each the investigated
populations were not completely identical and high gene
flow has occurred among the leaf rust population of dif-
ferent provinces. The highest differentiation and genetic
distance among the Iranian leaf rust populations was de-
tected between leaf rust population in Sistan and Balu-
chistan in South east followed by Golestan and Kerman-
shah provinces in North and Southwest, respectively.
Highest similar virulence pattern of physiological races
was observed between the rust population between
Ardabil province and other studied provinces followed
Copyright © 2013 SciRes. AJPS
Molecular Genetic Diversity in Iranian Populations of Puccinia triticina, the Causal Agent of Wheat Leaf Rust 1385
by the leaf rust population in Northern Khorasan. The
maximum number of virulence factor for 31 Lr genes
was found for P. t races TKTTN and TFTTN that were
collected form Ardabil and North Khorasan, respectively
[4]. The high pathogenic variability of leaf rust races in
Ardabil and Northern Khorasan may be indication that
these two regions are the center of origin of pathogenic
ariability and also the source of race distribution to the
other regions. Present and befor study [4] shows that leaf
rust population in Iran is highly dynamic and variable
and is subjected to frequent pathogenic shifts and there-
fore as long term strategy the wheat breeding programs
in Iran need to deploy combination of effective resistance
genes such as Lr9, Lr28, Lr25, Lr19, Lr29, and Lr2a with
non-race specific and adult-plant resistance genes of
which Lr34, Lr46, and Lr67 are slow rusting leaf rust resis-
tance genes widely used in breeding program worldwide.
During this process which may not be achieved in short
time, other control measures such as gene deployment,
and chemical control might need to be considered as
short term strategy.
The consistent results of molecular studies based on
phenotypic variation with pathogenicity in a very high
genetic diversity in Iranian leaf rust populations seems to
Iran is one of the centers of leaf rust diversity in the
world. Race diversity in Iran could be because the wheat
pathogen evolution probably in Iran is very old, since
Iran and its neighbors that are located in the Fertile
Crescent that be wheat Origins in the world and adjacent
with other regions of the world that wheat have been
identified as the primary origins and related to air flow
between the these regions, as well as very large popula-
tion, short cycle generation especially in epidemics con-
ditions that cause widespread epidemics in Iran and pri-
mary and alternative hosts are in different regions of Iran
probably all of the reasons for the racial diversity in Iran
be, so that they may be genetic structure of this fungus is
different from other populations in other parts of the
world. Oliveira and Samborski believed that the P.
triticina centre of origin is the Fertile Crescent region of
the Middle East, where the natural range of the primary
and alternative hosts overlaps [27]. It is believed that
origin of any plant where most of the existing genetic
diversity and so plants have evolved with disease.
5. Conclusion
Based on the results of leaf rust in Iran is very diverse
and has very high evolutionary potential, Thus stability
of single-gene resistance probably be short. The strategy
for breeding for resistance to this disease should be es-
tablished based on the use of quantitative genes (QTL)
with other resistance genes or race-non specific with race
specific resistance genes and Cultivars rotation, chemical
control and even rotation of use chemical fungicides.
Main strategy to control leaf rust disease in the country
should be use of a combination of genetic strategies and
fungicides. Effective resistance genes such as Lr9, Lr28,
Lr25, Lr19, Lr29 and Lr2a in combination with nonspecific
genes such as Lr34 and Lr46 or Lr67 could create a more
effective and stability resistance. Cultivars which have
different gene combinations are replaced and interchange
before the appearance of new pathogenicity phenotypes
will located, as rotation used in the resistance genes will
to stable and also cause long duration of the resistance
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