American Journal of Plant Sciences, 2013, 4, 1821-1833
http://dx.doi.org/10.4236/ajps.2013.49224 Published Online September 2013 (http://www.scirp.org/journal/ajps)
Phenotypic and Molecular Characterization of Wheat Leaf
Rust Resistance Gene Lr34 in Iranian Wheat Cultivars and
Advanced Lines
Seyed Taha Dadrezaei1,2*, Kumarse Nazari3, Farzad Afshari2, Ebrahim Mohammadi Goltapeh1
1College of Agriculture, TarbiatModares University, Tehran, Iran; 2Seed and Plant Improvement Institute (SPII), Karaj, Iran; 3Inter-
national Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria.
Received April 20th, 2013; revised May 20th, 2013; accepted June 15th, 2013
Copyright © 2013 Seyed Taha Dadrezaei 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.
ABSTRACT
Lr34 is a vital gene in developing resistance to leaf rust, stripe rust, and powdery mildew of wheat. Providing simulta-
neous resistance to various pathogens has made this gene valuable in breeding for wheat resistance to many diseases.
The present study investigates the csLV34 marker’s capability in diagnosing this locus in130 wheat commercial cultivars
and advanced wheat lines from Iran, and assesses the impact of this gene on disease severity in field conditions. To as-
sess the reactions of cultivars and lines which contained Lr34 under epidemic conditions of leaf rust, these cultivars were
cultivated during the 2009 and 2010 cropping season. Of the 130 studied cultivars, 43 contained Lr34. Cultivars that
were selected and studied in stress conditions had the most frequent presence of Lr34. It can be concluded that this gene
plays a vital role in increasing the tolerance of cultivars under stress conditions. Lr34 seems to cause active transition of
materials out of the cell. In addition to being resistant to several important diseases of wheat, Lr34 can increase tolerance
to stresses such as salinity. Considering the calculated value for AUDPC (3% - 440%/d) in cultivars containing Lr34, it
seems that some cultivars contained additional resistance genes. The rate of infection in all cultivars, when presence of
Lr34 was detected through the molecular marker, was lower than in other cultivars. Field results confirmed the results of
the analysis using the csLV34b molecular marker.
Keywords: Lr34 Gene; AUDPC; Salinity Stress; Leaf Rust; Puccinia triticina
1. Introduction
Leaf rust caused by Puccinia triticina (Pt) is the most
common and widely distributed of the three wheat rusts.
Although losses from leaf rust are usually less damaging
than those from stem rust and stripe rust, because of its
regular and widespread occurrence, the global leaf rust
damages are greater than the other two rust [1,2]. Wheat
leaf rust is present in all wheat growing areas in Iran. In
general, leaf rust is the second most important disease of
wheat in Iran but in southern areas leaf rust is the most
important disease of wheat [3]. The Pt population in Iran
is extremely dynamic and a large number of races were
found in a recent study of pathogenic variability of Pt
population in Iran [4]. Improving the resistance of wheat
cultivars to this disease is a preventive strategy with the
greatest effect on reducing its damage. Rust resistance
genes in wheat can be categorized into two groups: seed-
ling resistance genes and adult plant resistance (APR)
genes. Seedling resistance genes appear in both the seed-
ling and adult plant stages and can be recognized; as a
result, they show resistance in all phenotypic stages.
Seedling resistance genes often lead to a hyper sensitive
reaction (cell death-HR) or to lignifications of the cell
membrane [5]. Adult plant gene resistance acts non-spe-
cifically on race pathogens in the adult plant stage, and
cultivars containing these genes are susceptible at seed-
ling stage and have various levels of comparative resis-
tance to disease at the adult plant stage [6]. This type of
resistance is called race non-specific gene resistance since
there is no relationship between host genes and pathogen
genes. Additionally, it provides resistance to all pathogen
isolates. Compared with susceptible plants, Lr34 resistance
has a longer period of infection with fewer and smaller
uredinial pustules at two weeks after infection [6,7].
*Corresponding author.

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1822
More than 70 leaf rust resistance genes have been iden-
tified, most of which are involved in race-specific cate-
gory and follow the boom and bust cycle due to the high
pathogenic variability of Pt population. Among the race
non-specific genes, the Lr34/Yr18 complex [7], Lr46 [8]
and Lr67 [9] are the most commonly used genes in global
wheat. The Lr34 gene was initially introduced as an APR
gene in cultivar “Frontana” [10], this gene encoding ATP
Binding Cassette (ABC) transporter[7], with the locus of
the gene on the short arm of wheat chromosome 7D [11].
With few exceptions, the race specific genes are asso-
ciated with a very short durability. There is increasing
interests in identification and development of race-non-
specific slow rusting genes which have been shown to be
more durable than race-specific genes [12,13]. So far leaf
rust pathogens have not been reported as virulent on Lr34
[7,14]. Lr34 is genetically linked with the stripe rust
adult-plant resistance gene Yr18, morphological marker
leaf tip necrosis (Ltn1) [15], and an adult-plant powdery
mildew resistance gene (Pm38) [16,17]. Tolerance to
Barley yellow dwarf virus (Bdv1) in Lr34 carrying culti-
vars is also reported [18]. The incorporation of leaf rust
resistance gene Lr34 into “Thatcher” background is
known to enhance stem rust resistance [10,11]. These si-
multaneous resistances to several pathogens have made
the Lr34/Yr18 locus one of the most valuable gene regions
for disease resistance breeding in wheat. Leaf tip necrosis
(Ltn1) has been used as a phenotypic marker for field
selection of slow-rusting resistance conferred by Lr34/
Yr18 [15] by national and international breeding programs,
but because of variable expression of Ltn1 under differ-
ent environmental conditions and in different genetic
background [18], the leaf tip necrosis could not be used
as a reliable and diagnostic marker.
Development and application of molecular markers for
Lr34/Yr18 have been an important objective in marker-as-
sisted selection in breeding for durable leaf rust resis-
tance. Application of previously developed markers, such
as gwm295 and gwm1220 [19,20], has shown their lim-
ited use in breeding application due to their low diagnos-
tic capability in precise detection of Lr34 in different ge-
netic backgrounds. During the last few years significant
progress has been made in the development of more
closely linked markers for Lr34/Yr18 complex trait such as
SWM10 and csL V 34 [21]. More recently, Kolmer et al.
(2008) [18] confirmed robustness of a tightly linked
csLV34 marker with Lr34/Yr18 across a wide range of
global wheat germplasm and its utility in wheat breeding.
In present study seedling and adult-plant assessment of
resistance to leaf rust coupled with application of the
tightly linked marker csLV34 with Lr34/Yr18 were used in
the characterization of adult-plant resistance of some of
the Iranian bread and durum wheat genotypes to leaf rust.
2. Materials and Methods
2.1. Plant Materials
There were 130 commercial wheat cultivars and ad-
vanced lines of hexaploid and tetraploid wheat from Iran
(Table 1). Seeds of test genotypes were obtained from
department of cereal research at Seed and Plant Improve-
ment Institute Research (SPII), Karaj, Iran. A set of That-
cher near isogenic lines (TcNILs) were used both at seed-
ling and adult-plant assessments. Seeds of TcNILs were
kindly provided by International Maize and Wheat Im-
provement Center (CIMMYT). The universal leaf rust
susceptible cultivar “Thatcher” and a local susceptible
cultivar “Bolani” were used in seedling and adult plant
tests.
2.2. Seedling Test
Assessment of seedling resistance was carried out at ce-
real rust pathology laboratory at SPII. The 130 test ge-
notypes and leaf rust Thatcher near isogenic lines were
used in seedling assessment against a local Pt isolate.
Eight to 10 seeds of each genotype were planted in a 9
cm diameter pot filled with potting mix at two replica-
tions. The seedlings were grown in a rust-free green-
house at 20˚C and 16 hours light. At two leaf stage, seed-
lings were inoculated with the local leaf rust isolate col-
lected from the field trial site at Khuzestan Agricultural
Research Station, in south of Iran. This isolate used in
seedling tests and field inoculations. Urediniospores stored
at −80˚C were first heat shocked at 42˚C for 5 minutes
and then mixed with Talcum powder (1:4). Seedlings
were inoculated with the talc-spore mixture using a small
duster. Inoculated plants were placed overnight in a hu-
mid chamber at 17˚C ± 2˚C and dark condition. After the
incubation period, plants were placed in a greenhouse
with 20˚C ± 2˚C and 16 hrs supplementary light. Seed-
ling infection types were recorded 12 days post-inocu-
lation using; (fleck) and 0 to 4 scale [22]. Infection types;
and 0 to 3 were considered resistant reactions, while in-
fection types higher than 3 were considered as suscepti-
ble.
2.3. Field Experiments
In order to evaluate adult-plant resistance of test geno-
types and TcNILs, a field trial was carried out under mist
irrigation system at Khuzestan Agricultural Research
Center in 2009. Each genotype was planted as two 1-m
row plot and 30 cm space. To facilitate inoculum build-
up and uniform dissemination of infection, the suscepti-
ble cultivar Bolani was planted perpendicular to the rows
of entries. Bolani was also planted at each 10 plot inter-
vals. Disease epidemic was created by artificial inocula-

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1823
ger UV (Gel DocTM XR Bio rad Universal Hood II) was
used for visualization and documentation of banding pat-
terns.
tion of the local leaf rust isolate collected from the same
site in 2008. Preserved urediniospores were first multi-
plied on susceptible cultivar Bolani under greenhouse
conditions following the above described procedure. Fre-
shly collected urediniospores were mixed with talcum
powder and inoculation was carried over entries after
misting irrigation at late afternoons by atomizer back-
pack duster. First inoculation was started at 20 January
2010 when plants were at tillering stage and it was re-
peated four times at fortnight intervals. Disease severities
(0% - 100%) were recorded according to the Modified
Cobb’s scale [23] and the adult-plant reactions were re-
corded for the major infection types R (resistant), MR
(moderately resistant), MS (moderately susceptible) and
S (susceptible) according to Roelfs et al. (1992) [24].
Field scoring started from early onset of uniform infec-
tions in Bloani with 10 days intervals. Data on the dis-
ease severities and infection types were used in calcula-
tion of coefficient of infection (CI) for each individual
score [24].The area under the disease progress curve
(AUDPC) [25] was then calculated as follows using the
CIs for three disease scores:
3. Results
3.1. Molecular Marker Screening
The presence of a 150-bp band is diagnostic of the Lr34
gene, indicating the presence of Lr34 in cultivars and lines
carrying the gene. This band belongs to the csLV34b al-
lele, which is associated with Lr34. Another longer band
(229-bp) is produced and belongs to the csLV34a allele,
which is associated with the absence of Lr34; i.e. when
the cultivar does not contain Lr34, and then the longer
band is produced. However, in cultivars that are het-
erozygous both alleles are present and so both bands are
generated simultaneously.
Of the130 investigated genotypes, 87 lacked Lr34;
shown by the presence of the 229-bpband of allele
csLV34a. There were 43 genotypes with Lr34, shown by
the presence of the 150-bp band. These 43genotypes
were divided into two groups: 26 homozygous cultivars
with the 150-bp band and17 genotypes with both the
229- and 150-bp bands. Of the 17 hetero zygous geno-
types, 15 had unique phenotype markers. In these 15 ge-
notypes, there produced PCR product possessed a three-
band pattern consisting csLV34 a and csLV34b alleles,
both of which were accompanied by an additional band
with a higher molecular weight (280 bp). In the study of
global wheat cultivars Kolmer et al. (2008) observed the
three-band pattern in heterozygous cultivars; however,
most heterozygous cultivars had a two-band pattern and
cultivars with a three-band pattern were less frequent.
Moreover, the positive control sample in the present study
was the 150-bp band; and “Thatcher”, susceptible “Bo-
lani”, and all susceptible cultivars only produced the 229-
bp band and the negative control sample (water) had no
bands (Figure 1).

i1
N
ii1
i1 i
i1
yy
AUDPCt t
2









with:
i—index for scoring date;
yi—Coefficient of leaf rust infection at scoring date i;
ti—scoring date i expressed in days after scoring date 1;
n—total number of scoring dates in the trial.
2.4. DNA Extraction and PCR Analysis
For extraction of genomic DNA, 100 mg of harvested
leaves from 14 days old seedlings of each tested geno-
type was ground to fine powder on liquid nitrogen. The
fine powder was immediately transferred into a 2 ml
tubes and the small scale DNA extraction protocol was
followed as described in CIMMYT applied molecular
protocol [26]. PCR reaction was performed in 20 μl for
the CsLV34 marker following published protocol [18,21]
in a PTC 100 Thermocycler (MJ Research, Waltham,
MA). The PCR product was separated on 1.5% agarose
gel containing TBE 0.5× buffer. Digital molecular ima-
There was a high frequency of csLV34 allele (150 bp),
which is associated with Lr34 in the assessed Iranian lines
and cultivars. Of the 130 studied genotypes, 43 contained
the diagnostic Lr34 allele, representing a frequency of
33% (Table 1) as follows:
csLV34b (229 bp)
csL
V
34b (150 bp)
1 2 3 4 5 6 7 8
Figure 1. Polymerase chain reaction amplification products from wheat cultivars using csLV34 marker. 1. Thatcher +Lr34
(Lr34); 2. Falat (Lr34); 3. Ghods (Lr34); 4. Star (heterozygote); 5. Bam (Lr34); 6. Dez (Lr34); 7. Aflak (Lr34); 8. Cham ran (Lr34).

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1824
Table 1. Verification of Lr34 by applied molecular marker csLV34 and comparison of leaf rust infection in Iranian commer-
cial wheat cultivars and advanced lines in adult and seeding plant.
Entry
No Wheat variety Pedigree Final field score
Seedling
infection type AUDPC csLV34
1 Chamran Attila,(CM85836-50Y-OM-OY-3M-OY) Bread
Wheat 10MS 2 44 a
2 Falat Kvz/Buho"s"//Kal/Bb= Seri82 Bread
Wheat 5MS 0 22 a
3 Maron Avd*Pchu((28mt54A*N10-Brv21-
1c/Kt54B)Nar59,1093))7c
Bread
Wheat 30MS 2 290 a
4 Navid (Kirkpinar 79) 63-112/66-2*7C Bread
Wheat 40MS 3 340 a
5 Hirmand Byt/4/Jar//Cfn//Sr70/3/Jup"s" Bread
Wheat 5MS ; 23 b
6 Alvand 1-27-6275/CF1770 Bread
Wheat 60MS 3 580 a
7 Alamoot KVZ/Ti71/3/Maya"s"//Bb/Inia/
4/Kj2/5/Anza/3/Pi/Ndr//Hys
Bread
Wheat 30MS N , 1 220 a
8 Mahdavi Ti/Pch/5/Mt48/3/Wt*//Nar59/
Tota63/4/Mus
Bread
Wheat 50MS 2 560 a
9 Zarin PK15841 Bread
Wheat 5MS ; , 1 23 a
10 Darab 2 Maya"s"/Nac Bread
Wheat 10MS ; , 1 44 b
Check Susceptible
control Bolany Bread Wheat 80S 1120 a
11 Chanab Chanab Bread
Wheat 40MS 3 414 a
12 Tajan Bow"s"/Nkt"s"(CM67428-GM-LR-
5M-3R-LB-Y)
Bread
Wheat 20MS N , 1 90 b
13 Atrak Bow"s"/Nkt"s"(CM67428-GM-LR-
5M-3R-LB-Y)
Bread
Wheat 30MS 0 282 b
14 Nicknejad F13471/Crow"s" Bread
Wheat 30MS 3- 129 b
15 Kavir Stm/3/Kal//V534/Jit716 Bread
Wheat 30MS 2 , 1 202 a
16 Shirodi Attila,(CM85836-4Y-OM-OY-
8M-OY-opz)
Bread
Wheat R 0 4 a
17 Marvdasht HD2172/Bloudan//Azadi Bread
Wheat 5MS 0 24 a
18 Pishtaz Alvand//Aldan/Ias58 Bread
Wheat 20MS 2 242 a
19 Shiraz Gv/D630//Ald"s"/3/Azd Bread
Wheat 50S 3 510 a
20 Dez Kauz*2/Opta//Kauz Bread
Wheat 10MR 0 41 b
Check Susceptible
control Bolany Bread Wheat 90S 1410 a
21 Hamon Falat/Roshan Bread
Wheat 80S 3+ 660 a
22 Toos "Spn/Mcd//Cama/3/Nzr" Bread
Wheat 50S 3 572 a
23 Shahriar KVZ/Ti71/3/Maya"s"//Bb/Inia/4/
Kj2/5/Anza/3/Pi/Ndr//Hys
Bread
Wheat 50MS 2 250 a
24 Ghods Rsh/5/Wt/4/Nor10/K54*2//Fn/3/Ptr/
6/Omid//Kal/Bb
Bread
Wheat 60S 2 630 a

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines 1825
Continued
25 Sistan Bank"s"/Vee"s" Bread
Wheat 30MS 3 202 b
26 Bam Vee"s"/Nac//1-66-22 Bread
Wheat 30MS 2 202 b
27 Neishabour 1-63-31/3/12300/Tob//cno/sx Bread
Wheat 30MS 3- 200 b
28 Bahar Bloyka Bread
Wheat 10MS ; , 1 42 a
29 Moghan 1 (LR-N10B)*An3E Bread
Wheat 30MS 3 282 b
30 Moghan 2 chotiLerma Bread
Wheat 30MS 2 , 1 200 b
Check Susceptible
control Bolany Bread Wheat 70S 1010 a
31 Moghan 3 Luan/3/V763.23/V879.C8//Pvn/4/
Picus/5/Opata
Bread
Wheat 20MS ; 120 a
32 Darya SHAU/Chil Bread
Wheat 20MS 1 , 2 400 a
33 Yavarous Yavaros79 Durum
wheat 5MR 1 10 MIS
34 Zagros CN,79/7/2*Seri82 Bread
Wheat R 1++ 3 b
35 Arta Arta Bread
Wheat 20MS ; 161 a
36 Sepahan Azd/5/L2453/1347/4/Kal//Bb/Kal/3/
Au//Y50E/Kal*3
Bread
Wheat 20MS N , 1 160 b
37 Star Star"s" Bread
Wheat 20MS 2+ 120 b
38 Dena Tarro3 Durum
wheat R ; 3 MIS
39 Pishgham Bkt/90-Zhong87 Bread
Wheat 70MS 3 530 a
40 Sabalan 908*FnA12)*1-32-4382 Bread
Wheat 50MS 3 620 a
Check Susceptible
control Bolany Bread Wheat 90S
1310 a
41 Sivand Kaus"s"/Azd Bread
Wheat 10MR 2 40 a
42 Omid Omid Bread
Wheat 50MS 3 540 a
43 Shapasand Shapasand Bread
Wheat 90S 3 1130 a
44 Karaj 1 (200H*Vfn)Rsh Bread
Wheat 80S 3 1040 a
45 Karaj 2 (Fa*Th-Mt)Omid Bread
Wheat 70S 3 990 a
46 Karaj 3 (Drc*Mxp/Son64*Tzpp-Y54)Nai60 Bread
Wheat 60MS 3 580 a
47 Rasool Veery"s"=Kvz/Buho "s"//Kal/BB Bread
Wheat 40MS ; 440 b
48 Tabasea Tabasea Bread
Wheat 80S 3 1040 a
49 Adl Adl Bread
Wheat 50S 3 710 a

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1826
Continued
50 Inia Inia Bread
Wheat 40MS 1 402 b
Check Susceptible
control Bolany Bread Wheat 100S 1280a
51 Golestan Alondra”s” Bread
Wheat 20MS 2 161 a
52 Alborz Fn-Md*K117a/Cofn2(Son64-k1.Rend/
Cno”s”LR642-SON64)CM-2182
Bread
Wheat R N , 1 81 a
53 Kaveh Fta-P1 Bread
Wheat 50S 3 590 a
54 SorkhTokhm SorkhTokhm Bread
Wheat 80S 3 1160a
55 Azar 2 Azar 2 Bread
Wheat 40MS 2 482 a
56 Morvaread ilan/shaw7 Bread
Wheat R ; 3 a
57 Gaspard Gaspard Bread
Wheat 10MS 1+ 46 a
58 Gascogen Gascogen Bread
Wheat 20MS 2+ 122 a
59 Sayvan Sayvan Bread
Wheat 40MS 2 490 a
60 MV-17 MV-17 Bread
Wheat R ; 3 b
Check Susceptible
control Bolany Bread Wheat 70S 1150a
61 Karkheh Shwa/Mald//Aaz Durum
wheat R 1 3 MIS
62 Arya Stork Durum
wheat 20MS 2 160 MIS
63 N-88-3 MERUA//TURACO/CHIL/3/TAJAN Bread
Wheat 30MS 2 160 a
64 N-86-4 MILAN CM75118//KA CM 75118/
K1/3/TAJAN (DH)
Bread
Wheat 20MR ; 80 a
65 N-86-6 VORONA/CNO79//KAUZ/3/MILAN Bread
Wheat R 0
3 a
66 N-86-11 CMH82A.1294/2*KAUZ//MUNIA/
CHTO/3/MILAN
Bread
Wheat R 0 3 a
67 N-87-4 BAV92/PRINIA//TAM200/PRL Bread
Wheat R 0 3 a
68 N-87-6 JIMAI36/3/3/OASIS/SKAUZ//
4*BCN/4/89ZHONG2
Bread
Wheat 10MR 0 40 a
69 N-87-9 SUNSU/PBW343 Bread
Wheat R 0 3 a
70 N-87-13
PF74354//LD/ALD/4/2*BR12*2/3/
JUP//PAR214*6/FB6631/5/
SW89-5124*2/FASAN/6/TILH
Bread
Wheat R 0 3 a
Check Susceptible
control Bolany Bread Wheat 80S 1060a
71 N-87-16
NANJING2149/KAUZ/4/JUP/ALD”S”//
KIT”S”/3/VEE”S”/5/SHA 7//
HAHN”S”*2/PRL”S”
Bread
Wheat 30MS 3 160 a
72 C-87-14 SHA 7//HAHN”S”*2/PRL “S”/3/ATRAK Bread
Wheat 10MR 0 62 a
73 C-83-7 Alvand//Ns732/Her Bread
Wheat 40MS 2 402 a

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines 1827
Continued
74 C-83-8 130L1.11//F35.70/Mo73/4/Ymh/Tob
//Mcd/3/Lira
Bread
Wheat 10MS 2 122 b
75 C-84-8 Mihan = BKT/90Zhong87 Bread
Wheat 50MS 0 560 a
76 C-85-6 Mv17/Zrn Bread
Wheat 30MS 0 122 b
77 C-85-3 Ghk"S"/Bow"S"//90Zong87/3/Shiroodi Bread
Wheat 30MS 0 202 a
78 C-86-3 Bloudan/3/Bb/7C*2//Y50E/3*Kal/4/Mv17 Bread
Wheat 40MS 2 420 a
79 C-86-5 Yan7578.128//Chil/2*Star Bread
Wheat 40MS 3 340 b
80 C-86-6 Yan7578.128//Chil/2*Star Bread
Wheat 40MS 3 242 a
Check Susceptible
control Bolany Bread Wheat 80S 1020 a
81 M-85-6 Seri 82//Shuha"S"/4/Rbs/Anza/3/Kvz/
Hys//Ymg/Tob
Bread
Wheat 10MS 2++ 122 b
82 M-85-7 Seri82//Shuha"S"/4/Rbs/Anza/3/Kvz/
Hys//Ymg/Tob
Bread
Wheat R 3 4 b
83 M-85-15 Mv22-77//Stephon/3/Mon"s"/Lmu"s"//
Falke/4/Zarin
Bread
Wheat 50S 3 690 a
84 M-85-16 PASTOR/3/VORONA/CNO79//KAUZ Bread
Wheat R ; 4 a
85 M-85-16 PASTOR/3/VORONA/CNO79//KAUZ Bread
Wheat R ; 3 a
86 M-86-3 Gaspard/3/Jup/Bjy//Kauz/4/Kayson/Glenson Bread
Wheat 5MR 0 19 a
87 M-86-5 Alvd//Aldan/Ias*2/3/Gaspard Bread
Wheat 40MS 2+ 241 a
88 M-86-7 Alvd//Aldan/Ias/3/Druchamps/4/kauz/Stm Bread
Wheat 30MS 1 201 a
89 M-86-9 Owl 85256-*3OH-*O-*EOH/Mv17/
3/Alvd//Aldan/Ias
Bread
Wheat 40MS 3 401 a
90 M-87-18 BABAX/LR42//BABAX Bread
Wheat R ;
3 a
Check Susceptible
control Bolany Bread Wheat 60S 960 a
91 Aflak S-80-18 Bread
Wheat 5MR 3 12 b
92 S-83-3 Attila 50Y//Attila/Bcn Bread
Wheat R 0 3 a
93 S-83-4 F60314.76/MRL//CNO79/3/KA/NAC/
4/STAR
Bread
Wheat R 0 3 b
94 S-84-14 PASTOR/3/KAUZ*2/OPATA//KAUZ Bread
Wheat 10MS ; 48 b
95 S-85-19 INQALAB91*2/KUKUN Bread
Wheat R ; 3 a
96 S-87-2 VEE/PJN//2*KAUZ/3/PASTOR Bread
Wheat 5MR 0 12 a
97 S-87-8 KAUZ*2/BOW//KAUZ/3/BABAX Bread
Wheat 10MR 3 22 a
98 S-87-12 PASTOR/3/VORONA/CNO79//KAUZ Bread
Wheat 5MR ; 12 a

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1828
Continued
99 S-87-18 CBRD-3/STORK X DICOCCOIDES Bread
Wheat 5MR ; 12 a
100 S-87-20 OASIS/SKAUZ//4*BCN/3/2*PASTOR Bread
Wheat 30MS ; 122 b
Check Susceptible
control Bolany Bread Wheat 70S 1070 a
101 S-87-21 520- BABAX/LR42//BABAX*2/
3/VIVITSI
Bread
Wheat R 0 3 a
102 DM-79-2 PORTO-7 Durum
wheat 5MR 0 16 MIS
103 DM-81-6 PLATA-1/SNM//PLATA-9 Durum
wheat R ; 3 MIS
104 DM-82-6 SOOTY-9/RASCON-37 Durum
wheat R ; 3 MIS
105 DM-83-10 AUK/GUIL//GREEN Durum
wheat R ; 3 MIS
106 DM-84-3 RASCON-37/BEJAH-7 Durum
wheat R ; 4 MIS
107 DM-85-10 RASCON-37/BEJAH-7 Durum
wheat 5MR ; 12 MIS
108 WS-85-10 PRL/2*PASTOR Bread
Wheat 10MR 3 22 a
109 WS-85-15 PBW343*2/KONK Bread
Wheat R 0 3 b
110 WS-86-5 Shi#4414/Crow"S"//Azd Bread
Wheat R 0 3 a
Check Susceptible
control Bolany Bread Wheat 80S 1080 a
111 WS-86-8 SW89.5181/KAUZ Bread
Wheat R 0 25 b
112 WS-86-11 MUNIA/3/RUFF/FGO//YAV79/4/
PASTOR
Bread
Wheat 20MS ; 83 a
113 WS-86-12 PJN/BOW//OPATA*2/3/CROC_1/
AE.SQUARROSA (224)//OPATA
Bread
Wheat 10MS 0 42 b
114 WS-86-13 VORONA/CNO79//KAUZ/3/MILAN Bread
Wheat 5MS 0 22 a
115 WS-86-14 KAUZ/PASTOR Bread
Wheat 20MS 0 82 b
116 MS-85-15 Ombu/Alamo//Mahooti/3/1-66-22 Bread
Wheat 30MS 0 124 b
117 MS-85-12 Ombu/Alamo//Alvd/3/Kauz/Stm Bread
Wheat 30MS 1 160 b
118 MS-84-13 GF-gy54/Attila Bread
Wheat 40MS 3 242 b
119 MS-85-17 Sakha 8/Darab#2//1-66-22 Bread
Wheat 30MS 3 204 b
120 MS-84-16 Gkzombor/Zrn Bread
Wheat 30MR 3 300 a
Check Susceptible
control Bolany Bread Wheat 80S 1300 a
121 SS-85-6 1-66-22/3/GUP/BGY//kauz Bread
Wheat 30MS 3 202 b
122 SS-85-10 OMBU/ALAMO//ALVD/3/1-66-22 Bread
Wheat 30MS 3 202 b

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1829
Continued
123 SS-85-11 OMBU/ALAMO//MAHOOTI/3/1-66-22 Bread
Wheat 30MS ; 202 b
124 SS-85-14 SAKHA 8/DARAB#2//1-66-22 Bread
Wheat 30MS ; 282 b
125 SS-85-20 OMBU/ALAMO//KAV/3/PASOR/
SORKHTOKHM..
Bread
Wheat 30MS 0 202 b
126 DW-79-5 LAGOST-2 Durum
wheat 20MS ; 89 b
127 DW-81-18 SORA/2*PLATA12 Durum
wheat 20MS ; 85 b
128 DW-84-5 GREEN_14//YAV_10/AUK Durum
wheat R ; 6 MIS
129 Bezostaya Bezostaya Bread
Wheat 3 b
130 Veerynak Veerynak Bread
Wheat 2 a
Check Susceptible
control Bolany Bread Wheat 100S 3+ 1280 a
Check Positive
control Thatcher (Lr34) Bread
Wheat 30MS 3- 300 b
Among commercial Iranian cultivars, 18 of 64 culti-
vars contained csLV34b, indicating presence of Lr34; only
“Hirmand” and “Bam” which were the result of national
crossing program, which had origin in international germ-
plasm.
None of the nine advanced lines bread wheat for the
warm and humid climate of northern Iran carried Lr34, i.e.
they contained csLV34a. Among the nine advanced lines
bread wheat for cold climates, three had Lr34; among the
11 advanced lines bread wheat for the warm climate of
the south, five carried Lr34.
From nine lines bread wheat for mild climates, two
had the 150-bp band indicating the presence of Lr34.
However, cultivars studied in environmental stress con-
ditions and selected to assess their tolerance of these
conditions were totally different as follows. Among the
eight chosen advanced lines bread wheat for humid stress
conditions, four had Lr34 (50%). Of the five selected ad-
vanced lines bread wheat for saline conditions located in
mild climate areas, four contained Lr34; among five ad-
vanced lines bread wheat for saline conditions in the
warm climate of the south, all five carried Lr34, i.e. 90%
of advanced cultivars bread wheat to tolerate salinity
contained Lr34. It seems that cultivars carrying Lr34 could
better tolerate stress conditions, and most cultivars pre-
viously introduced for areas with salinity stress in mild
climates such as “Hirmand”, “Sistan”, “Neishabour”,
“Nicknejad”, “Tajan”, and “Darab2” contained Lr34.
“Inia” is a cultivar resistant to salinity in experiments and
also contained Lr34. “Star” is a late maturity cultivar, and
is presently widely cultivated in Khuzestan Province. In
evaluation of salinity resistance of wheat cultivars in
laboratory and field conditions, the percentage of germi-
nation and seedling establishment of “Star” under saline
conditions was good relative superiority. “Bam” was re-
cently introduced for mild climate areas with soil and
water salinity stress, and contains Lr34 [27]. Cultivars that
were not resistant to environmental stress such as salinity
lacked Lr34, e.g. lines bread wheat for the northern cli-
mate or cultivars “Darya”, “Golestan”, “Alborz”, “Kaveh”,
and “Bahar” (Table 1).
In the present research, 13 genotypes of durum wheat
were also investigated, four cultivars and seven lines of
which did not produce any bands to confirm the presence
or absence of Lr34. A separate experiment for these cul-
tivars was repeatedly conducted with positive and nega-
tive controls and a similar result was obtained. Absence
of a reproduced band or piece in durum wheat was likely
due to the lack of the D genome since Lr34 is located on
the small arm of chromosome 7 of genome D and prim-
ers should be placed on this part to be reproduced. Be-
cause of the absence of this genome in tetraploid culti-
vars (e.g. durum wheat), this piece was not reproduced in
these cultivars.
3.2. Phenotypic Characterization
About 44 cultivars gave R or MR reactions in field as-
sessment, among which only eight carried Lr34. Most
cultivars (35 genotypes) contained Lr34 or were MS. The
estimated AUDPC for cultivars carrying Lr34 was within
3% - 440%/day, indicating that some cultivars may carry

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1830
some other resistance genes as well as Lr34. This was
confirmed by further studies conducted with other mark-
ers of race-specific genes on the same cultivars (unpub-
lished data). In wheat cultivars with a combination of
resistance genes genetic infection type with the highest
resistance conceals the impact of the type with lower in-
fection; therefore, these cultivars that contain race-spe-
cific resistance genes in addition to Lr34, an infection
type of R or MR is seen instead of MS resistance type
and the presence of Lr34 is masked by other main genes.
Accordingly, methods such as molecular methods which
can easily identify this gene are important. The pheno-
typic method used currently relies on Ltn1 and makes it
very difficult to recognize Lr34 from the visual phenotype
of leaves since Ltn1 does not express equally in different
environments. This method requires a lot of experience
and the results are not always correct.
AUDPC for the examined cultivars in the present
study was within 3% - 1410%/day. AUDPC of control
susceptible “Bolani”, which was repeated 13 times among
the field-grown cultivars (was planted at the end of the
experiment and after each plot of 10 cultivars as suscep-
tible control), was calculated to be 960% - 1410%/d, this
difference in AUDPC is the result of environment.
AUDPC of other cultivars, except for that of the sus-
ceptible control (Check), was 3% - 1160%/day. The lower
AUDPC belonged to cultivars that were resistant due to
their effective race-specific resistance genes and were
discussed previously. The results showed that AUDPC of
500% - 800%/day indicated a susceptible cultivar and
AUDPC > 800 indicated a too susceptible one.
In this experiment AUDPC < 500 was regarded as ac-
ceptable resistance because about two months after an epi-
demic of the disease and at the time of maximum flag
leaf efficiency in photosynthesis and grain filling, the
maximum infection remained at 40 MS. The highest
AUDPC for genotypes containing Lr34 was for “Rasool”,
“Inia”, and line C-86-5 with values 440%, 402%, and
340%/day, respectively. Most lines and cultivars con-
taining this gene had AUDPC of about 200%/day and
“Thatcher” had 300%/day. Accordingly, AUDPC of 250
- 500 was considered as semi-susceptible or relatively re-
sistant; AUDPC of 150 - 250 was considered semi-re-
sistant, and AUDPC < 150 was considered resistant.
The flag leaf plays a crucial role in grain filling. The
surface of this leaf in susceptible cultivars can be rapidly
covered with leaf rust pustules at the time of grain filling.
As a result, the entire surface can be infected and so
harms its function. However, cultivars containing Lr34
are resistant to rapid development of the pathogen and
delay it. The flag leaf of such cultivars is more capable of
grain filling and incurs less damage.
In epidemics, leaf rusts do much damage to flag leaves;
therefore, assessing resistance to leaf and yellow rust at
the adult plant stage is very important in improvement
programs. Most assessment of resistance to leaf rust is
done on the flag leaf because severity of the disease on
leaves reflects the primary growth of the pathogen and
damage to the plant [24].
An obvious advantage of presence of Lr34/Yr18 in cul-
tivars is the absence of high intensities of infection at the
end of the wheat growing season. However, cultivars that
do not contain this gene can be highly infected by leaf
rust during the whole growing season. Cultivars with
race-specific genes, which are widely used in cultivars,
are expected not to show long-term resistance for patho-
types that are virulent on the Lr9 resistance gene. These
were previously discussed by Kolmer [28]. However, if
resistance of a race-specific gene is broken, Lr34 prevents
rapid epidemics of the disease and major damage. In all
cultivars in which the presence of Lr34 was shown by
molecular marker, infection rate was less than in culti-
vars not containing this gene (Table 1). Field results con-
firmed the analysis results concerning the csLV34b mar-
ker (Table 1).
Lr34 is believed to be dominant. The results clearly
showed gradual rust resistance in heterozygous cultivars
and with no difference for cultivars homozygous for this
gene.
Cultivars not containing Lr34 included 87 genotypes,
divided into four groups according to resistance and sus-
ceptibility in field and greenhouse as follows.
The first group of seven genotypes was susceptible in
the seedling stage and resistant in the adult plant stage:
N-87-16, C-86-6, M-86-9, S-87-8, WS-85-10, and MS-
84-16. These genotypes are crucial since they carry a
gene or genes of adult plant stage resistance other than
Lr34. Markers are needed to verify and identify the pres-
ence of these genes.
The second group included 57 genotypes resistant in
both adult plant and seedling stages. This group con-
tained race-specific resistance genes to the utilized genes.
These genotypes may contain non-specific race resis-
tance genes other than Lr34 that are masked by the effect
of specific resistance genes.
The third group included 17 cultivars which were sus-
ceptible in both seedling and adult plant stages. This
group lacked adult plant stage genes and effective spe-
cific race genes to the applied isolate.
In the fourth group, five cultivars were resistant or
immune in the seedling stage but susceptible in the adult
plant stage. This showed that these cultivars lacked adult
plant stage resistance genes; however, they were influ-
enced by pathogen races other than those present in the
seedling stage in the field. At the time of collecting
spores and testing them in greenhouse condition, this

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines 1831
race did not exist or was just part of the field’s patho-
genic population. The population or race which could in-
fect these cultivars was not present in the population ga-
thered and used in the greenhouse, or alternatively their
frequency was low. Thus, they did not have the opportu-
nity to appear under greenhouse conditions but could
have greater effect in the field due to the longer time
available.
Cultivars carrying Lr34 were categorized into three
groups, based on their reaction to leaf rust in field and
greenhouse. Cultivars in the first group included 33 ge-
notypes that were resistant in both seedling stage and
adult plant stages, indicating that they contained effective
race-specific genes other than Lr34.
The second category included two groups. The first
group contains delight cultivars susceptible in the seed-
ling stage and semi-resistant (MR) or semi-susceptible
(MS) in the adult plant stage. This is characteristic of
Lr34 and these cultivars apparently carried only Lr34. The
second group was “Aflak” and the line M-85-7. They
were totally susceptible in the seedling stage and com-
pletely resistant in the adult plant stage. It seems that
these cultivars lack the effective race-specific resistance
gene to the utilized isolate. However, their high resis-
tance in field conditions indicated that they contained a
gene or genes of adult plant stage resistance other than
Lr34. This makes them unique and they require further
investigation.
4. Discussion
In the present study, in cultivars of Iranian origin Lr34
was only present in cultivars and lines linked with the
very old 22-66-1 lines of ill-defined pedigree. Almost all
other Iranian cultivars containing this gene originated
from international germplasm, especially from CYMMIT.
Lr34was also present in cultivars of international germ-
plasm. Introducing cultivars of CYMMIT origin into Iran
has increased the frequency of Lr34in Iranian cultivars.
Lr34 has received much attention in recent years, since
this gene is present in high frequency in CIMMYT bread
wheat germplasm and derived cultivars with CIMMYT
origin [29].
Examination of 123 local cultivars (landraces) of Iran
by Kolmer et al. [18] showed that only three cultivars
(2.4%) had Lr34. Also, in other parts of the world, the
csLV34b allele did not exist in most local cultivars and
had a low frequency, compared with the general fre-
quency in improved wheat cultivars. The incongruity of
csLV34b occurring among improved and local cultivars
may be directly or indirectly caused by improvement
trials to combine Lr34/Yr18 into new cultivars. Among
international cultivars, those from CYMMIT showed high
frequency (30%) of csLV34b.
The mentioned result was verified by assessing the in-
fection, analysis of molecular markers, and data gathered
through pedigree for presence of Lr34. Of 130 cultivars,
which had Lr34 according to pedigree, specific bands
were produced in only 43. Due to the high sensitivity of
this marker in detecting the Lr34 gene allele, having pure
seeds from the desired cultivars, and not mixing with
other cultivars are critical to providing reliable results.
Studies have shown that due to probable mistakes in data
in pedigrees, it is crucial to apply specific molecular mar-
kers to confirm the presence of resistant genes against
leaf rust in wheat cultivars. Many researchers have con-
cluded that molecular markers are better for this predic-
tion than pedigree data [30,31].
In the present study, cultivars selected and investigated
in stress conditions had the highest percentage presence
of Lr34 and it seems that this gene was effective in in-
creasing the tolerance of cultivars in environmental stress
conditions. All chosen lines and cultivars of the warm
and humid climate in the north of Iran lacked Lr34, and
these cultivars were selected in environmental conditions
without stresses such as drought, heat, cold, and salinity.
The warm and dry climate of the south of Iran, followed
by lines bread wheat lines for cold climates, had the
highest frequency of Lr34.
Lr34 belongs to the super family of ABC transporters
that produce proteins connected to the plasma membrane,
which plays an important role in transferring materials in
and out of the membrane. ABC transporters can transport
a wide range of materials that can be cytotoxic, including
ions, so that they transport macromolecules against the
diffusion gradient on both sides of the cell membrane
[32,33]. Drug transporters were primarily recognized in
cancer cells which were resistant to drugs. These trans-
porters carry the consumed drugs out of cancer cells and
make the cells resistant to drugs. This mechanism was
also discovered in drug-resistant fungi such that, in the
resistant mutant fungi, gene expression or drug trans-
porter genes and accordingly related proteins greatly in-
creased. Consequently, by discharging more and lower-
ing fungicide concentrations below the fatality threshold
in fungal cells this causes resistance to fungicides. In ad-
dition to fungicide disposal, drug transporters can pass
mycotoxin discharge of other fungi, natural antimicrobial
compounds of other organisms, and plant defense com-
pounds out of the cell and cause resistance in fungi [34].
It seems that ABC transporters are one effective factor
in resistance to salinity in plants. This system is probably
active in cultivars resistant to salinity that contain ABC
transporters, and extra salt ions are actively pumped out
of the cells. As a result, transporters enable salinity tol-
erance in various cultivars or help the process of identi-

Phenotypic and Molecular Characterization of Wheat Leaf Rust Resistance Gene Lr34
in Iranian Wheat Cultivars and Advanced Lines
1832
fying salinity ions and prevent them entering the cytosol.
Accordingly, these cultivars are probably resistant to sa-
linity. This hypothesis was also strengthened by assess-
ing durum cultivars, which lack the D genome and are
more susceptible to salinity than wheat cultivars. There-
fore, Lr34—in addition to providing resistance to leaf rust,
yellow rust, powdery mildew, and barley yellow dwarf
virus in wheat—plays an important role in improving
tolerance to environmental stresses such as salinity. Per-
haps one mechanism of Lr34 in providing relative resis-
tance to leaf rust agent pathogen is removing toxins, me-
tabolites, or other harmful substances discharged by pa-
thogens into host cells. As an example, virulent factors,
which are discharged by haustoria of pathogens and are
vital for aiding pathogen growth in the host cell, are
pumped out of the cell by these transporters. This makes
the pathogen grow slowly compared to hosts which lack
this gene. Additionally, the phenotype of fewer and
smaller pustules, and a longer latent period, would con-
sequently appear since these substances lower the growth
of the out -of-cell pathogen and reduce its density in the
environment. This material is harmful to plant cells and
its removal causes the plant tolerance to increase with no
negative impact on plant physiological functions. Pump-
ing harmful substances out of plant cells actually in-
crease the plant’s tolerance to biotic and abiotic stresses.
A more accurate assessment of the relationship between
this gene and the tolerance to environmental stresses
such as salinity needs a further and more complete inves-
tigation.
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