Plant Pathol. J. 31(4) : 402-413 (2015)
http://dx.doi.org/10.5423/PPJ.OA.12.2014.0127
pISSN 1598-2254 eISSN 2093-9280
The Plant Pathology Journal
©The Korean Society of Plant Pathology
Research Article Open Access
Resistance Potential of Bread Wheat Genotypes Against Yellow Rust Disease
Under Egyptian Climate
Amer F. Mahmoud1*, Mohamed I. Hassan2 and Karam A. Amein2
Department of Plant Pathology, Faculty of Agriculture, Assiut University, Assiut, Egypt
2
Department of Genetics, Faculty of Agriculture, Assiut University, Assiut, Egypt
1
(Received on December 15, 2014; Revised on June 27, 2015; Accepted on July 5, 2015)
Yellow rust (stripe rust), caused by Puccinia striiformis
f. sp. tritici, is one of the most destructive foliar diseases
of wheat in Egypt and worldwide. In order to identify
wheat genotypes resistant to yellow rust and develop
molecular markers associated with the resistance, fifty
F8 recombinant inbred lines (RILs) derived from a
cross between resistant and susceptible bread wheat
landraces were obtained. Artificial infection of Puccinia
striiformis was performed under greenhouse conditions
during two growing seasons and relative resistance index (RRI) was calculated. Two Egyptian bread wheat
cultivars i.e. Giza-168 (resistant) and Sakha-69 (susceptible) were also evaluated. RRI values of two-year trial
showed that 10 RILs responded with RRI value > 6 < 9
with an average of 7.29, which exceeded the Egyptian
bread wheat cultivar Giza-168 (5.58). Thirty three RILs
were included among the acceptable range having RRI
value > 2 < 6. However, only 7 RILs showed RRI value
< 2. Five RILs expressed hypersensitive type of resistance (R) against the pathogen and showed the lowest
Average Coefficient of Infection (ACI). Bulked segregant analysis (BSA) with eight simple sequence repeat
(SSR), eight sequence-related amplified polymorphism
(SRAP) and sixteen random amplified polymorphic
DNA (RAPD) markers revealed that three SSR, three
SRAP and six RAPD markers were found to be associated with the resistance to yellow rust. However, further molecular analyses would be performed to confirm
markers associated with the resistance and suitable for
marker-assisted selection. Resistant RILs identified in
the study could be efficiently used to improve the resis*Corresponding author.
Phone) +201010081105, FAX) +20882331384
E-mail) amer.mahmoud@agr.au.edu.eg
This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
tance to yellow rust in wheat.
Keywords : bread wheat, bulked segregant analysis (BSA),
yellow rust, molecular markers, Puccinia striiformis f. sp. tritici
Wheat is the most widely cultivated cereal crop in the
world. In Egypt, it is considered as the major winter cereal
crop and the third major crop in terms of area planted. Severe losses due to different wheat diseases including yellow
rust, also known as stripe rust, have been reported (Kissana
et al., 2003). Wheat yellow rust is one of the most devastating diseases of wheat worldwide. It is caused by the basidiomycete fungus Puccinia striiformis Westend. f. sp. tritici
Eriks (Eriksson, 1894; Hassebrauk, 1965; Stubbs, 1985 and
Hovmoller et al., 2010) and continues to cause severe damage worldwide (Chen et al., 2013). Biotrophic plant pathogens such as rust pathogens secrete an array of proteins,
known as effectors, to modulate plant innate immunity and
enable parasitic infection (Hogenhout et al., 2009). Yellow
rust is a highly destructive disease threatening wheat production and quality worldwide. This is mainly due to the
pathogen’s ability to mutate and multiply rapidly as well
as to use its air borne dispersal mechanism from one field
to another (Brown and Hovmøller, 2002; Watson and De
Sousa, 1983).
Slow rusting, a form of quantitative resistance, prolongs
the latent period of fungal infection and decreases disease
severity (Rashid, 1997; Wang et al., 2000), can slow the
incidence and development of stripe rust in the field, thus
reducing yield losses and being of practical value. Slow
rusting is thought to be a non-race-specific resistance that
effectively controls the epidemic spread of stripe rust in a
stable, sustained, and durable manner (Das et al., 1992).
Among all the control measures of this disease, genetic
resistance is the only economic and practical control measure, causing no additional cost to the farmer (Singh et al.,
2004). Therefore, breeding for resistance to yellow rust
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Resistance to Yellow Rust in Bread Wheat
and developing new resistant cultivars became the main
target in wheat breeding programs and considered as the
most economical and effective way to eliminate the use of
fungicides and reducing crop losses caused by the disease.
Afshari (2004) and Singh et al. (2004) reported that a
new stripe rust race can attack wheat cultivars which
possessing Yr27 resistance gene in India, Yemen, Egypt,
Ethiopia, Eritrea, Tajikistan, Uzbekistan and Kyrgyzstan
during previous years Thus, it is of great importance to
develop wheat cultivars possessing new resistance genes
for yellow rust.
A number of genes controlling yellow or stripe rust
resistance in wheat has been identified (McIntosh et
al., 2011). Moreover, genetic associations of various
microsatellite or simple sequence repeats (SSR) and
random amplified polymorphic DNA (RAPD) markers
with stripe rust resistance genes have been reported in
wheat (Akfirat et al., 2010; Bariana et al., 2002; Bariana
et al., 2006; Chague et al., 1999; Khlestkina et al., 2007;
Robert et al., 2000; Sun et al., 2002; Tabassum 2011;
Wang et al., 2002; William et al., 2003; Wang et al.,
2008). Identification of molecular markers associated
with yellow rust resistance has facilitated the markerassisted selection of the resistance genes in wheat
breeding program. Furthermore, to ensure optimal costeffectiveness, molecular markers used for marker-assisted selection should permit efficient screening of large
populations (Huang and Röder, 2004). Bulked segregant
analysis (BSA) is a highly efficient method developed
firstly by Michelmore et al. (1991) for rapidly identifying markers linked to any specific gene or genomic
region. The use of BSA in combination with PCR-based
markers such as RAPD, SSR and SRAP markers has
proven to be a very powerful technique for identifying
molecular markers associated with a quantitative trait
locus (QTL) or a gene of interest (Avila et al., 2003;
Bakhit and Abdel-Fatah, 2013 and El-Sayed et al., 2013;
Cho et al., 1996; Diaz-Ruiz et al., 2010; Nakamura et
al., 2001; Rostoks et al., 2002; Shen et al., 2003; Torres
et al., 2010).
In Egypt, a large number of wheat landraces have been
preserved, which potentially possess many yellow rust
resistance genes. Thus, it is of great importance to identify
resistance genetic resources from the landraces and use
them in wheat breeding programs aiming to develop improved varieties. In the present study, a population of fifty
F8 recombinant inbred lines (RILs) derived from a cross
between resistant and susceptible Egyptian bread wheat
landraces was used to identify genotypes resistant to yellow rust, and to develop molecular markers associated with
403
the disease resistance.
Materials and Methods
Plant material and greenhouse trials. The plant material utilized in the present study consisted of a population
of fifty F8 recombinant inbred lines (RILs) derived from a
cross between resistant and susceptible bread wheat landraces to yellow rust collected from farmers’ fields in Upper
Egypt in 1993. Two Egyptian bread wheat cultivars i.e.
Giza-168 (resistant) and Sakha-69 (susceptible) were used
as controls.
The trials pertaining to screening different genotypes for
their resistance against yellow rust were conducted at the
greenhouse of Plant Pathology Department, Faculty of Agriculture, Assiut University, Egypt during 2011 and 2012.
During this investigation, fifty two entries (fifty RILs and
two Egyptian wheat cultivars) were sown at the greenhouse
to observe the yellow rust response. A 6 seeds of each entry was planted in a sterilized pot (No.8) containing sterilized soil, supplemented with NPK at the ratio of 1%. Three
replicates were made for each genotype. The plants were
irrigated when necessary and daily observed for infection.
Collection of yellow rust samples and inoculation. Diseased leaf samples were collected from different cultivars
and breeding lines from three different locations in Assiut
Governorate, Egypt, namely Assiut, Manfalout and Abuteeg
during 2011 and 2012 wheat growing seasons. Artificial
inoculations were carried out using a spores mixture of the
most prevalent yellow rust of the field collections in 2011
and 2012. Diseased leaf samples were placed on moist
filter paper in a Petri dish that was kept at 10ºC overnight.
Urediniospores were collected from diseased leaf samples
with the help of small brush. The inoculums were prepared
immediately prior to use by suspending urediospores in a
solution of diH2O and Tween-20, which 1 to 2 drops were
added to break the surface tension. Spore suspension was
prepared 0.6 ml per plant at concentration of 6 × 105 spores/
ml. Spray were apply on the plants on front and back from
6 inch away with one hand behind the plants to catch most
of the inoculum on the plant. Inoculations were carried
out in the early evening (after sunset).The inoculation of
all plants was carried out at booting stage according to the
method of Tervet and Cassell (1951).
Disease observation. Observations were recorded at the
first appearance of stripe rust infection on the susceptible
wheat lines. Observations on response and severity of
stripe rust were recorded according to Loegering (1959)
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Mahmoud et al.
Table 1. The observation on response of stripe rust
Reaction
No Disease
Resistant
Resistant to Moderately Resistant
Moderately Resistant
Moderately Resistant to Moderately
Susceptible
Moderately Susceptible
Moderately Susceptible to Susceptible
Susceptible
Fig. 1. Scale of rust severity (percent of leaf area infected).
and Hussain (1997). Yellow rust severity (%) was recorded
for each cultivar from the time of rust first appearance then
every seven days until the early dough stage (Large, 1954).
Estimates of severity were measured according to Modified Cobb Scale (Paterson et al., 1948), which is used to
determine the percentage of possible tissue rusted and was
evaluated from 1% to 100%. The severity was recorded as
percent of rust infection on the plants (Fig. 1). As severity
is determined by visual observation, readings cannot be
absolutely correct. Therefore, below 5% severity, the intervals used are trace (T) to 2. Usually, 5 percent intervals are
used from 5 to 20 percent severity and 10 percent intervals
for higher readings.
Readings of severity and reaction are recorded together
with severity first as follow:
TR = Trace severity of resistant type infection
10MR = 10 percent severity of a moderately resistant
type infection
30MS = 30 percent severity of a Moderately Susceptible
type infection
50S = 50 percent severity of a susceptible type infection
Calculation for ACI and RRI. The Coefficient of Infection (CI) for stripe rust has been calculated according to
(Akhtar et al., 2002) as shown in Table 1. Coefficient of
Infection was calculated by multiplying the response value
with the intensity of infection in percent. Average Coefficient of Infection (ACI) was derived from the sum of CI
values of each entry divided by the number of tested years.
The highest ACI of a candidate line is set at 100 and all
other lines are adjusted accordingly. This gives the Country
Average Relative Percentage Attack (CARPA). The ‘0’ to
Observation
Response
value
O
R
RMR
MR
0.0
0.2
0.3
0.4
MRMS
0.6
MS
MSS
S
0.8
0.9
1.0
‘9’ scale previously designated as Resistance Index (R.I)
has been re-designated as RRI (Relative Resistance Index).
From CARPA, RRI was calculated on a 0 to 9 scale, where
0 denotes most susceptible and 9 denotes highly resistant
(Akhtar et al., 2002). The RRI was calculated according to
the following formula:
Bulked segregant analysis (BSA). In order to identify
molecular markers associated with the resistance against
yellow rust in specific genomic regions, the F8 RILs population was subjected to BSA with three molecular marker
systems including simple sequence repeat (SSR), sequencerelated amplified polymorphism (SRAP) and random
amplified polymorphic DNA (RAPD) markers. The BSA
was performed at the Department of Genetics, Faculty of
Agriculture, Assiut University in 2013.
Based on RRI value of the two-year trial for each RIL,
seven resistant and seven susceptible contrasting genotypes
selected from the RILs population were used to construct
resistant and susceptible DNA bulks. DNA extraction
from young and fresh leaves of each RIL was carried
out according to the cetyltrimethylammonium bromide
(CTAB) method for isolation of total genomic DNA from
plants (Murray and Thompson, 1980) with some modifications. Aliquots of DNA from the two extreme groups of
seven resistant and seven susceptible RILs were mixed to
produce resistant and susceptible DNA bulks for BSA.
Molecular markers analysis. Resistant and susceptible
DNA bulks were screened for differences using sixteen 10mer RAPD primers (Operon, USA), eight SSR and eight
SRAP makers. Primers sequences and PCR conditions of
SSR markers were obtained by the GrainGenes database
(http://wheat.pw.usda.gov). PCR amplifications were per-
2015-11-26 오후 1:24:04
Resistance to Yellow Rust in Bread Wheat
formed in 25 μl reaction mixtures, each containing 50-100
ng of genomic DNA, 1× PCR buffer, 2-4 mM MgCl2 (2
mM for SSR and 4 mM for RAPD and SRAP), 200 μM
of each dNTP, 0.2 μM of each primer, and 1 U Taq DNApolymerase. Amplifications were performed in a SensoQuest LabCycler (SensoQuest GmbH, Göttingen, Germany) using the following PCR profile: initial denaturation at
94oC for 5 min, followed by 45 cycles each consisting of 1
min at 94oC, 1 min at 34oC for RAPD, 40-45oC for SRAP
and 50-55oC for SSR (depending on the suggested annealing temperature), followed by 2 min at 72oC, with a final
extension at 72oC for 10 min. PCR products were separated
using horizontal gel electrophoresis unit on 1.5% agarose
gels for RAPD and 2.5% for SSR and SRAP in 0.5 × TBE
buffer. A 100 bp DNA ladder was used to estimate the size
of each amplified DNA fragment. The gel was run for approximately 2-3 hrs using constant voltage of around 80 V
and then visualized and photographed under UV light. Putative polymorphisms among the two bulks were detected
for each marker separately.
Results
In the present study, a total number of fifty F8 RILs derived
405
from a cross between resistant and susceptible bread wheat
landraces, and two Egyptian bread wheat cultivars i.e.
Giza-168 (resistant) and Sakha-69 (susceptible) were evaluated for yellow rusting resistance under artificial infection
at Plant Pathology Department, Faculty of Agriculture,
Assiut University, Egypt. The following parameters were
used to assess yellow rusting at both tested years: Disease
Reaction (DR), Coefficient of Infection (CI), Average Coefficient of Infection (ACI) and Relative Resistance Index (RRI).
Disease reaction (DR). Of 50 RILs, 3 (6%) RILs (i.e. RIL3, RIL-4 and RIL-15) showed Susceptible (S) symptoms
(Average Coefficient of Infection 90-100%), 4 (8%) RILs
(i.e. RIL-7, RIL-13, RIL-14 and RIL-17) showed Moderately Susceptible (MS) symptoms (ACI 80- < 90%), 17
(34%) RILs showed Moderately Susceptible to Susceptible (MSS) symptoms (ACI: 60- < 80%), 12 (24%) RILs
showed Moderately Resistant to Moderately Susceptible
(MRMS) symptoms (ACI: 40- < 60%), 5 (10%) RILs
showed Resistant to Moderately Resistant (RMR) symptoms (ACI: 30- < 40%), 4 (8%) RILs (i.e. RIL-27, RIL-28,
RIL-44 and RIL-47) showed Moderately Resistant (MR)
symptoms (ACI: 20- < 30%) and 5 (10%) RILs (i.e. RIL31, RIL-32, RIL-42, RIL-43 and RIL-48) were Resistant (R)
Table 2. Response of fifty RILs and two Egyptian bread wheat cultivars (Giza-168 and Sakha-69) to yellow rust infections during
2011and 2012
Genotypes
RIL-1
RIL-2
RIL-3
RIL-4
RIL-5
RIL-6
RIL-7
RIL-8
RIL-9
RIL-10
RIL-11
RIL-12
RIL-13
RIL-14
RIL-15
RIL-16
RIL-17
RIL-18
RIL-19
RIL-20
RIL-21
Disease Reaction
2011
50MRMS
60MS
90MSS
90S
80MSS
60MS
80MSS
75MSS
80MSS
60MS
90MS
50MRMS
85MSS
80MSS
90S
70MS
90MSS
90MSS
70MS
75MS
50MRMS
2012
60MS
70MSS
100S
100S
90MSS
90MSS
90S
90MSS
85MSS
70MS
85MS
60MS
90S
90S
100S
80MS
90S
70MS
80MS
75MS
60MRMS
Coefficient of Infection
2011
2012
CI
Total
30
48
81
90
72
48
72
67.5
72
48
72
30
76.5
72
90
56
81
81
56
60
30
48
63
100
100
81
81
90
81
76.5
56
68
48
90
90
100
64
90
56
64
60
36
78
111
181
190
153
129
162
148.5
148.5
104
140
78
166.5
162
190
120
171
137
120
120
66
ACI
RRI
39
55.5
90.5
95
76.5
64.5
81
74.25
74.25
52
70
39
83.25
81
95
60
85.5
68.5
60
60
33
5.49
4.00
0.85
0.45
2.11
3.19
1.71
2.31
2.31
4.32
2.70
5.49
1.50
1.71
0.45
3.60
1.30
2.83
3.60
3.60
6.03
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406
Mahmoud et al.
Table 2. Continued
Genotypes
RIL-22
RIL-23
RIL-24
RIL-25
RIL-26
RIL-27
RIL-28
RIL-29
RIL-30
RIL-31
RIL-32
RIL-33
RIL-34
RIL-35
RIL-36
RIL-37
RIL-38
RIL-39
RIL-40
RIL-41
RIL-42
RIL-43
RIL-44
RIL-45
RIL-46
RIL-47
RIL-48
RIL-49
RIL-50
Giza-168
Sakha-69
Disease Reaction
2011
70MS
65MS
60MRMS
70MS
70MS
65MR
60MR
65MRMS
80MSS
45R
50MR
80MS
90MSS
70MS
70MS
75MSS
85MSS
75MS
70MS
55MRMS
40R
40MR
50MR
75MS
75MS
55MR
20R
70MS
60MRMS
70MS
80MSS
2012
65MS
75MS
80MS
65MRMS
70MRMS
60MR
55MR
75MS
80MS
40R
35R
75MS
80MS
75MS
80MSS
80MSS
85MS
75MS
70MS
60MRMS
50R
55RMR
60MRMS
70MS
80MSS
60MRMS
30R
65MRMS
65MRMS
60MRMS
90S
Coefficient of Infection
2011
2012
CI
Total
56
52
36
56
56
26
24
39
72
9
20
64
81
56
56
67.5
76.5
60
56
33
8
16
20
60
60
22
4
56
36
40
72
52
60
64
39
42
24
22
60
64
8
7
60
64
60
72
72
68
60
56
36
10
16.5
36
56
72
36
6
39
39
36
90
108
112
100
95
98
50
46
99
136
17
27
124
145
116
128
139.5
144.5
120
112
69
18
32.5
56
116
132
58
10
95
75
76
162
ACI
RRI
54
56
50
47.5
49
25
23
49.5
68
8.5
13.5
62
72.5
58
64
69.75
72.25
60
56
34.5
9
16.25
28
58
66
29
5
47.5
37.5
38
81
4.14
3.96
4.50
4.72
4.59
6.75
6.93
4.54
2.88
8.23
7.78
3.42
2.47
3.78
3.24
2.72
2.49
3.60
3.96
5.89
8.19
7.53
6.48
3.78
3.06
6.39
8.55
4.72
5.62
5.58
1.71
Legend:
R:
RMR:
MSS:
S:
Resistant
Resistant to Moderately Resistant
Moderately Susceptible to Susceptible
MR:
MRMS:
MS:
Moderately Resistant
Moderately Resistant to Moderately Susceptible
Moderately Susceptible
Susceptible
(ACI 5- < 20%). The Average Coefficient of Infection of
the two Egyptian bread wheat cultivars Giza-168 (resistant)
and Sakha-69 (susceptible) were 38 and 81%, respectively
(Table 2).
Relative resistance index (RRI). Frequency distribution of
RRI values of the two-year trial for 50 F8 RILs is presented
in Fig. 2. The distribution was continuous and approached
normality, indicating a quantitative type of inheritance, and
thereby RRI is under the control of multiple genes. Based
on the RRI values (Table 2), among the 50 tested RILs, 9
RILs (i.e. RIL-27, RIL-28, RIL-31, RIL-32, RIL-42, RIL43, RIL-44, RIL-47 and RIL-48) had expressed resistant (R)
to moderately resistant (MR) type of reaction. These genotypes were having highest relative resistance index (RRI)
of yellow rust resistance, which exceeded the resistant cultivar Giza-168 (5.58). The RRI for mentioned seven RILs
were: 6.75, 6.93, 8.23, 7.78, 8.19, 7.53, 6.48, 6.39 and 8.55
respectively (Fig. 3, Table 2). Maximum stripe rust severity was recorded in RIL-3, RIL-4 and RIL-15 with RRI of
0.85, 0.45, and 0.45, respectively, which was in magnitude
smaller than the susceptible wheat cultivar Sakha-69 (1.71)
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Resistance to Yellow Rust in Bread Wheat
407
Fig. 2. Frequency distribution of relative resistance index (RRI)
in a population of 50 F8 RILs evaluated during two years (where,
0 denotes most susceptible and 9 denotes highly resistant).
Fig. 4. DNA amplification patterns obtained using bulked segregant analysis with 8 SSR markers. M is the 100 bp DNA ladder, RB the resistant bulk, SB the susceptible bulk. Differences
between the two bulks were detected using Xgwm339-2AS,
Xgwm493-3BS and Xwmc398-6BS. Arrows indicate polymorphic bands obtained which distinguished the resistant from the
susceptible bulk.
Fig. 3. Histograms depicting RILs used to create: (A) resistant
and (B) susceptible bulks.
(Table 2).
Yellow rust severity (%). The yellow rust severity recorded for the susceptible RILs and the two Egyptian cultivars
estimated for two years (Table 2) revealed that, none of the
investigated RILs having (0%) severity. Among the 50 tested RILs, yellow rust severity in 2011 was maximum (90%)
for RIL-3, RIL-4, RIL-11, RIL-15, RIL-17, RIL-18 and RIL34; and minimum (20-50%) for RIL-1, RIL-12, RIL-21, RIL31, RIL-32, RIL-42, RIL-43, RIL-44 and RIL-48.
In 2012, the maximum severity for the tested RILs was
(90-100%) in RIL-3, RIL-4, RIL-5, RIL-6, RIL-7, RIL-8,
RIL-13, RIL-14, RIL-15, RIL-17 and Sakha-69. The minimum yellow rust severity was (30-50%) in RIL-31, RIL32, RIL-42 and RIL-48.
Bulked segregant analysis (BSA). For identification of
molecular markers associated with yellow rust resistance,
resistant and susceptible DNA bulks were screened for
differences using sixteen 10-mer RAPD primers (Operon,
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Mahmoud et al.
Table 3. Polymorphism detected between resistant and susceptible bulks by the use of three SSR, three SRAP and six RAPD markers
Marker
Sequence (5′–3′)
Xgwm339-2AS
F: AATTTTCTTCCTCACTTATT
R: AAACGAACAACCACTCAATC
F: TTCCCATAACTAAAACCGCG
R: GGAACATCATTTCTGGACTTTG
F: GGAGATTGACCGAGTGGAT
R: CGTGAGAGCGGTTCTTTG
Xgwm493-3BS
Xwmc398-6BS
Total
Average
SRAP-2
SRAP-3
SRAP-5
ME7-F: TAGGTCCAAACCGGACC
EM8-R: GACTGCGTAGCAATTACT
ME7-F: TAGGTCCAAACCGGACC
EM9-R: GACTGCGTACGAATTAGA
ME8-F: TGAGTCCAAACCGGACT
EM9-R: GACTGCGTACGAATTAGA
Total
Average
OPA-13
OPC-07
OPC-18
OPG-10
OPG-11
OPK-17
CAGCACCCAC
GTCCCGACGA
TGAGTGGGTG
AGGGCCGTCT
TGCCCGTCGT
CCCAGCTGTG
Total
Average
USA), eight SSR and eight SRAP makers.
SSR markers: Out of eight SSR markers tested, three
SSRs (37.5%) namely Xgwm339, Xgwm493 and Xwmc398
located on chromosomes 2AS, 3BS and 6BS, respectively,
distinguished resistance from susceptible bulks (Fig. 4)
and generated a total number of 39 bands ranged from 9
(Xgwm493-3BS) to 16 (Xwmc398-6BS) with an average
of 13 bands per marker. Of the 39 bands amplified with 3
SSRs, 17 bands (43.6%) were polymorphic (Table 3) with
an average of 5.7 polymorphic bands per marker. The lowest
polymorphism between the two bulks (22.2%) was obtained
with Xgwm493-3BS, whereas the highest polymorphism
(64.3%) was produced with Xgwm339-2AS (Table 2).
SRAP markers: Among the eight SRAP markers screened,
three (37.5%) SRAPs showed polymorphism between
resistance and susceptible bulks (Fig. 5). The three markers generated a total of 22 bands with an average of 7.3
bands per marker which ranged from 6 bands for SRAP-
Bands
amplified
Polymorphic
bands
Polymorphism
(%)
14
9
64.3
9
2
22.2
16
6
37.5
39
13
17
5.7
43.6
9
4
44.4
7
2
28.6
6
2
33.3
22
7.3
8
2.7
36.4
13
10
11
17
14
13
2
2
1
7
3
2
15.4
20
9.1
41.2
21.4
15.4
78
13
17
2.8
21.8
5 to 9 bands for SRAP-2 (Fig. 5, Table 3). Of these 22
bands generated with three SRAPs, 8 bands (36.4%) were
polymorphic with an average of 2.7 polymorphic bands per
marker. The highest polymorphism (44.4%) was observed
with SRAP-2, whereas 28.6% and 33.3% polymorphism
was obtained by SRAP-3 and SRAP5, respectively.
RAPD markers: Out of 16 RAPD primers tested, 6 RAPD
primers (37.5%) showed polymorphic amplification patterns which distinguished resistance from susceptible bulks
(Fig. 6). Individual primers produced bands in a range of
10 (OPC-07) to 17 (OPG-10), with an average of 13 bands
per marker. Of the 78 bands amplified with 6 primers, 17
bands (21.8%) were polymorphic (Table 3) with an average
of 2.8 polymorphic bands per marker. Polymorphic bands
for individual primers ranged from a unique band (9.1%),
which was present only in the resistance bulk, with OPC18 to 7 bands (41.2%) with OPG-10. These bands could be
considered as specific markers for yellow rust resistance in
wheat.
2015-11-26 오후 1:24:06
Resistance to Yellow Rust in Bread Wheat
409
Fig. 5. DNA amplification patterns obtained using bulked segregant analysis with 8 SRAP markers. M is the 100 bp DNA ladder, RB the resistant bulk, SB the susceptible bulk. Differences
between the two bulks were detected using SRAP-2, SRAP-3
and SRAP-5. Arrows indicate polymorphic bands obtained which
distinguished the resistant from the susceptible bulk.
Discussion
Tolerance to yellow rust is one of the most important objectives of wheat breeding programs in all wheat growing
regions of the world (Akfirat et al., 2010). Wheat landraces
from diverse geographic regions are a potential source of
novel rust resistance genes for developing new and diverse
resistant germplasm (Sthapit et al., 2014). A large number
of wheat landraces have been preserved in Egypt, which
possessed abundantly genetic diversity including many
yellow rust resistance genes. Therefore, it is of great importance to identify resistance genetic resources from the
landraces to be used in wheat breeding programs aiming to
develop improved varieties. In the present study, a popula-
Fig. 6. DNA amplification patterns obtained using bulked segregant analysis with 16 RAPDs markers. M is the 100 bp DNA ladder, RB the resistant bulk, SB the susceptible bulk. Differences
between the two bulks were detected using OPA-13, OPC-07,
OPG-10, OPG-11, OPK-17 and OPC-18. Arrows indicate polymorphic bands obtained which distinguished the resistant from
the susceptible bulk.
tion of fifty F8 RILs derived from a cross between resistant
and susceptible Egyptian bread wheat landraces was evaluated for yellow rust resistance under greenhouse conditions
during two growing seasons. RRI values of two-year trial
(Table 2) revealed that, among the 50 tested RILs, 5 RILs
(RIL-31, RIL-32, RIL-42, RIL-43 and RIL-48) expressed
2015-11-26 오후 1:24:06
410
Mahmoud et al.
hypersensitive type of resistance against the pathogen and
4 RILs (i.e. RIL-27, RIL-28, RIL-44 and RIL-47) had expressed moderately resistant type of reaction. These genotypes were having highest RRI which exceeded the resistant cultivar Giza-168, suggesting that resistant genotypes
are expected to possess diverse resistance genes and could
be efficiently used as parents to improve resistance to yellow rust in breeding programs.
Bulked segregant analysis in combinations with three
molecular markers systems revealed that out of eight SSR
and eight SRAP and sixteen RAPD markers tested, three
SSR, three SRAP and six RAPD markers distinguished
resistance from susceptible bulks and were found to be putatively associated with the resistance to yellow rust. Therefore, these markers could be useful as a base for markerassisted selection program aiming to improve yellow rust
resistance in wheat. In accordance with these results, genetic associations of various SSR and RAPD markers with
stripe or yellow rust resistance genes have been reported
in wheat (Akfirat et al., 2010; Bariana et al., 2002; Bariana
et al., 2006; Chague et al., 1999; Khlestkina et al., 2007;
Robert et al., 2000; Sun et al., 2002; Tabassum, 2011;
Wang et al., 2002; Wang et al., 2008; William et al., 2003).
When utilizing BSA in segregating populations with minimal gene distortion, the likelihood of falsely identifying
linked markers to the target gene is minimized; therefore,
fewer individuals are required per bulk (Lin et al., 2006).
Thus, BSA can provide fast detection of molecular markers
linked to genes of interest. RAPD analysis in combination
with BSA has been long used to identify molecular markers linked to genes of interest (Bakhit and Abdel-Fatah,
2013; Chague et al., 1997; Mackay and Caligari, 2000; Lin
et al., 2006; Michelmore et al., 1991; Zhang et al., 1994).
SRAP is an efficient molecular technique with the marker
behaves as codominant and more reproducible than RAPD
(Li and Quiros, 2001). However, unlike RAPD and SRAP,
SSRs reported firstly in plants by Condit and Hubbel (1991)
are PCR-based markers characterized by a high level of
polymorphism that permits to discriminate among cultivars
and even among closely related wheat breeding lines (Maccaferri et al., 2007; Mantovani et al., 2008). Moreover,
SSRs are locus-specific, codominant markers evenly distributed over the genome and require only small amounts
of genomic DNA for analysis which are particularly useful
for mapping and genetic analysis. A large number of SSR
markers are already available for several important agricultural crops including wheat (Gupta and Varshney, 2000;
Mantovani et al., 2008; Röder et al., 1998; Somers et al.,
2004; Sourdille et al., 2004; Wang et al., 2007). The 2013
Catalogue of Gene Symbols for Wheat (McIntosh et al.,
2013) and the 2013-2014 Supplement include 67 officially
named Yr genes (Yr1 to Yr67) designated for the resistance
to stripe rust and 42 with temporary Yr designations. Furthermore, over 140 QTLs for resistance to yellow rust in
wheat have been published and through mapping flanking
markers on consensus maps, 49 chromosomal regions are
identified (Rosewarne et al., 2013). In the present study,
we identified three SSR markers associated with the resistance to yellow rust in bread wheat; namely Xgwm339,
Xgwm493 and Xwmc398 located on chromosomes 2AS,
3BS and 6BS, respectively. Accordingly, many of previously reported Yr genes, QTLs and SSR markers associated
with the resistance to yellow rust in wheat were located
on chromosomes 2A, 3B and 6B (McIntosh et al., 2013;
Rosewarne et al., 2013). Chromosome 2A has one region
associated with the resistance on the short arm (2AS) and
another region on the long arm (2AL). The region around
the 2AS QTL (QRYr2A.1) are associated with the major,
race-specific seedling resistance gene Yr17 (Rosewarne
et al., 2013), while a major QTL for adult-plant resistance
(QYr.osu-2A) was located on chromosome 2AS by Fang
et al., 2011. The 3B chromosome appears to have at least
three regions associated with stripe rust resistance. The
majority of QTLs identified on 3B are on the short arm
(3BS) and it is interesting that Xgwm493 was mapped into
QRYr3B.1 region on 3BS which is known to be extremely
important as it is the location of Yr30 gene, and it has a
consistent intermediate effect on stripe rust and is fairly
consistent across environments (Rosewarne et al., 2013).
The temporarily designated gene Yrns-B1 on 3BS, which
identified as non-race specific adult-plant resistance gene
against stripe rust, was located in a 3 cM interval between
Xgwm493-3B and Xgwm1329-3B (McIntosh et al., 2013).
Recently, Zhou et al., 2014 reported that, the recessive gene
Yrwh2 is located on chromosome 3BS and it is different
from previously reported stripe rust resistance genes Yr30,
QYr.ucw-3BS, Yrns-B1, YrRub and QYrex.wgp-3BL previously mapped on chromosome 3B. They also suggested
that Yrwh2 and its closely linked markers are potentially
useful for developing stripe rust resistance wheat cultivars
if used in combination with other genes. Lowe et al., 2011
reported that the gene underlying QYr.ucw-3BS appears
to be different from Yr30 and Yrns-B1 genes. The QYrucw.3BS was mapped 3.6 cM distal of Xgwm493 by Lowe
et al., 2011, whereas the adult plant resistance gene YrnsB1 was mapped 2.5 cM proximal of Xgwm493 (Borner
et al., 2000; Khlestkina et al., 2007). However, Zhou et
al., 2014 could not determine the distance between Yrwh2
and Xgwm493 as the marker was not polymorphic. Similarly, chromosome 6A had three clearly defined regions
2015-11-26 오후 1:24:06
Resistance to Yellow Rust in Bread Wheat
associated with stripe rust resistance and these are likely
to be conferred by three distinct genes (Rosewarne et al.,
2013). The first region (QRYr6B.1) is at the telomere of
6AS. Generally, molecular markers tightly linked to target
genes can provide a useful tool in plant breeding since
they can used to detect tolerant genes of interest without
the need of carrying out field evaluations. Moreover, the
use of molecular markers can increase the efficiency of
conventional plant breeding by identifying markers linked
to the trait of interest which are difficult to evaluate and/
or are largely affected by the environment. Screening
a large number of breeding materials or populations at
early growth stages and in a short time could be also performed using molecular markers. Therefore, it is urgent
to identify new genes for resistance to yellow rust and to
develop molecular markers for efficient incorporation and
pyramiding of new genes into wheat cultivars (Zhou et
al., 2014). Consequently, SSR markers identified in the
present study (i.e. Xgwm339-2AS, Xgwm493-3BS and
Xwmc398-6BS) which showed the highest polymorphism
(43.6%) than SRAPs (36.4%) and RAPDs (21.8%) could
be efficiently used for marker-assisted selection of yellow
rust resistance in wheat breeding programs. Moreover, it
is likely that resistant RILs identified in the present study
contain potentially some adult plant resistance genes. For
example, the non-race specific adult-plant resistance gene
(Yrns-B1) which was identified previously as located on
3BS and linked to Xgwm493-3BS could be present within
the resistant RILs. Furthermore, some seedling and/or adult
plant resistance genes on 2AS and 6BS may also be present. In addition, diseased leaf samples were collected from
different cultivars and breeding lines across different locations, thereby there is a possibility of the presence of more
than one pathotype. Therefore, it is likely that interaction
between disease resistance genes could also be present.
Subsequently, resistant RILs identified in the current study
could be efficiently used to develop improved varieties
in wheat breeding programs. However, further molecular
analyses of the whole RIL population with co-segregating
markers would be performed to confirm markers associated
with the resistance and suitable for marker-assisted selection.
In conclusion, resistant RILs identified in the present
study are expected to possess diverse resistance genes and
could be efficiently used as parents to improve resistance
to yellow rust in wheat breeding programs. Molecular
markers identified in the study using BSA, once verified by
genotyping the whole RILs population, could allow implementation of marker-assisted selection for incorporating
desirable resistant genotypes in wheat breeding programs.
411
Furthermore, SSRs are likely to be the most effective markers used in the study.
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