Genetic dissection of quantitative powdery mildewresistance loci in tetraploid wheat

Roi Ben-David • Zvi Peleg • Amos Dinoor •

Yehoshua Saranga • Abraham B. Korol •

Tzion Fahima

Received: 16 July 2014 / Accepted: 7 October 2014 / Published online: 15 November 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Durum wheat, Triticum turgidum ssp.

durum Desf., is an important crop particularly in the

Mediterranean basin. Powdery mildew, caused by the

pathogen Blumeria graminis f. sp. tritici (Bgt), is a

major disease of wheat that results in significant yield

losses worldwide. A recombinant inbred line (RIL)

population, derived from a cross between durum

wheat and wild emmer wheat, T. turgidum ssp.

dicoccoides, was used for genomic dissection of

quantitative and qualitative resistance loci against

wheat powdery mildew based on a genomic map of

[600 markers, evenly distributed across the A and B

genomes of tetraploid wheat. The genetic analysis of

the phenotypic reactions of the RIL population to two

Bgt isolates revealed two different resistance mecha-

nisms. The first is monogenic: a wild emmer wheat

allele in a single locus conferring complete resistance

to Bgt#15, previously designated as PmG16. The

second one is polygenic: a set of durum wheat alleles,

in five independent QTLs that control partial resis-

tance to Bgt#66 in the RIL population, with a LOD

score range of 3.4–19.8. One of them is a major

quantitative resistance locus (QRL) that was mapped

on chromosome 1A and explains 26.4 % of the

variance. In most of the detected QRLs, the durum

wheat alleles conferred resistance to powdery mildew.Electronic supplementary material The online version ofthis article (doi:10.1007/s11032-014-0178-0) contains supple-mentary material, which is available to authorized users.

R. Ben-David

Institute of Plant Sciences, Agricultural Research

Organization (ARO)-Volcani Center, Bet Dagan 5025000,

Israel

e-mail: rbendav@sci.haifa.ac.il

Z. Peleg � Y. Saranga

The Robert H. Smith Faculty of Agriculture, Food and

Environment, The Robert H. Smith Institute of Plant

Sciences and Genetics in Agriculture, The Hebrew

University of Jerusalem, Rehovot 7610001, Israel

A. Dinoor

Department of Plant Pathology and Microbiology,

The Robert H. Smith Faculty of Agriculture, Food and

Environment, The Hebrew University of Jerusalem,

Rehovot 7610001, Israel

A. B. Korol � T. Fahima (&)

Institute of Evolution and the Department of Evolutionary

and Environmental Biology, Faculty of Natural Sciences,

University of Haifa, 199 Abba-Hushi Avenue,

Mount Carmel, Haifa 3498838, Israel

e-mail: fahima@research.haifa.ac.il

123

Mol Breeding (2014) 34:1647–1658

DOI 10.1007/s11032-014-0178-0

These findings are exceptional in the sense that, so far,

only a few Pm alleles originated from a durum wheat

background. Therefore, our results emphasize the high

potential of exploiting the wide genetic diversity of

tetraploid wheat germplasm for wheat breeding using

modern wheat genomics tools.

Keywords Blumeria graminis f. sp. tritici � Durum

wheat � Powdery mildew � QRL � Quantitative

resistance �Wild emmer wheat

Introduction

Wheat (Triticum spp.) is one of the world’s most

important food crop, which provides about one-fifth of

the calories and proteins consumed by humans

(FAOstat 2012, http://www.faostat.fao.org). Durum

wheat [T. turgidum ssp. durum (Desf.) MacKey.]

(2n = 4x = 28, genome BBAA) is the second most

widespread Triticum species constituting 10–11 % of

the world’s wheat cultivation land and accounts for

about 5 % of total wheat production. The Mediterra-

nean basin, close to the initial wheat domestication

and cultivation region, is producing most (*75 %) of

the world’s durum grains, but durum wheat is an

important crop also in other wheat-growing regions

(Elias and Manthey 2005). Durum wheat is the main

source for production of pasta, couscous, burghul and

other Mediterranean local end-products (Nachit et al.

2001). The ever-increasing human population con-

comitantly with loss of agricultural land (due to

urbanization processes, industrialization, desertifica-

tion and climatic changes) and diminishing resource

availability pose serious challenges to world agricul-

ture. To feed the 9 billion people expected by 2050

(http://www.fao.org/wsfs/world-summit/en/), a sig-

nificant grain yield increase of approximately 44

million metric tons per year would be needed (Tester

and Langridge 2010). It is estimated that pathogens

can cause potential yield loss of 16–23 % with greater

losses in intensive production agri-systems (Oerke

2006). Developing new cultivars with improved dis-

ease resistance is an economically and environmen-

tally safe approach to reduce yield losses. This

solution, however, requires a comprehensive explo-

ration of potential genetic resources and an in-depth

understanding of their resistance mechanisms.

Powdery mildew, caused by the biotrophic patho-

gen Blumeria graminis (DC.) E.O. Speer f. sp. tritici

Em. Marchal (Bgt hereafter), is a foliar wheat disease

resulting in severe yield losses worldwide. B. graminis

is an obligate biotrophic fungus of the order Erysip-

hales (Ascomycota), family Erysiphaceae (Braun et al.

2000). The continuous threat for breakdown of race-

specific resistance to powdery mildew (e.g., Bennett

1984; Hsam and Zeller 2002) is forcing a consecutive

effort to uncover new genes and alleles from the

resistance gene reservoir. Studies conducted in the last

75 years to identify powdery mildew resistance in

durum wheat germplasm yielded only a few resources

(reviewed by Bennett 1984). A single dominant gene,

designated Mld, was identified in durum wheat cv

Yuma (Briggle 1960). This powdery mildew resis-

tance gene was combined with Pm2 to produce

resistance in a British spring wheat cultivar. This

resistance has broken down due to a matching

virulence gene in mildew populations (Bennett

1984). A phenotypic test for resistance to powdery

mildew in 92 wheat lines resulted in only one durum

line possessing resistance (Upadhyay et al. 1972).

Screening of 2,500 exotic durum wheat and 500 exotic

bread wheat lines facilitated the detection of 35

additional resistant durum lines (Bahadur et al. 1977,

1979(. The allele Pm3h and the gene PmDR147 were

both derived from durum wheat (Hsam and Zeller

2002; Zhu et al. 2004).

Several genetic maps of crosses between durum

wheat and wild emmer have been constructed (e.g.,

Peng et al. 2000; Peleg et al. 2008; Ji et al. 2008) and

used for identification and allocation of various

agronomically important traits in tetraploid wheat,

including powdery mildew resistance genes (e.g.,

Ben-David et al. 2010; Ji et al. 2008). Qualitative

disease resistance is under simple genetic control and

is well characterized. By contrast, other forms of

disease resistance controlled by genetically complex

systems are less understood (Tanksley 1993). Most

complex resistance traits are controlled by multiple

loci, and their phenotypes are measured quantitatively.

Young (1996) defined the genetic loci associated with

this kind of resistance as quantitative resistance loci

(QRLs). This term is frequently used synonymously

with the term ‘‘partial resistance loci.’’ The number of

QRLs tends to range from one to ten or more in any

one system, suggesting that the number of loci

involved in quantitative disease resistance (QDR)

1648 Mol Breeding (2014) 34:1647–1658

123

might generally be lower than for other agriculturally

important traits (e.g., growth rate, biomass production,

fertility) (Young 1996). The identification of QRLs

plays a major role in crop improvement implemented

through marker-assisted selection (Miedaner and

Korzun 2012) and provides the genetic basis for

positional cloning of resistance genes.

A recombinant inbred line (RIL) population

derived from a cross between durum wheat and its

direct progenitor, wild emmer wheat [T. turgidum ssp.

dicoccoides (Korn.) Thell.], was used to genetically

dissect grain yield and yield components (Peleg et al.

2009a, b, 2011) as well as mapping of a single

dominant powdery mildew resistance gene, desig-

nated as PmG16 (Ben-David et al. 2010). In the

current study, we used the same population to: (1)

characterize the quantitative response to a Bgt isolate,

which is virulent on PmG16; (2) determine the

chromosomal location and phenotypic effects of QRLs

associated with wheat response to powdery mildew;

and (3) assess the novelty of the new QRLs compared

to known powdery mildew QRLs.

Materials and methods

Plant and fungal materials

A population of 152 F7 RILs was developed using the

single-seed descent (SSD) approach, from a cross

between durum wheat (cv. Langdon, LDN hereafter)

and wild emmer wheat (accession G18-16) (Peleg

et al. 2008). This RIL population was subjected to a

quantitative phenotypic analysis after inoculation with

Bgt isolate #66, collected from a natural population of

wild emmer wheat in Ammiad, northern Israel

(32�560N, 35�320E; 290 m above sea level). This

isolate is part of the ‘‘Eshed-Dinoor mildew collec-

tion,’’ maintained at the Hebrew University, and is

virulent on PmG16 that segregates in the same RIL

population (Ben-David et al. 2010).

Phenotypic characterization

A qualitative phenotypic test was performed at the

seedling stage on detached leaf segments, as previ-

ously described in Ben-David et al. (2010). The

quantitative response to Bgt#66 was characterized by

counting the number of powdery mildew pustules per

cm2 at the seedling stage on detached leaf segments at

three different days post-inoculation (DPI) points.

Leaf segments of the tested lines were maintained in

polystyrene boxes with water agar (6 g agar L-1)

supplemented with 50 mg L-1 of benzimidazole. The

detached leaves were infected with powdery mildew

spores in a settling tower at inoculum density of *500

spores cm-2. The leaf segments were incubated at

15 �C with a 12-h photoperiod of white fluorescent

light (photosynthetic photon flux density of

55–65 lmol m-2 s-1). The density of the deposited

spores per cm2 and the proportion and density of

germinated spores were determined in each of the

polystyrene boxes (six random measurements per box)

24 h after inoculation using a light microscope.

Optimization of the quantitative phenotypic assay

was performed, and it is detailed in the supplementary

section (see Fig. 1S and additional text). Inoculation

of the complete RILs population was performed on

two independent dates (90 days apart). These two

dates serve as two separate environments in the

ANOVA and QRL analysis.

The two parental lines (LDN and G18-16) were also

characterized for reaction to Bgt#66 at the adult plant

stage (Zadoks index 57; Zadoks et al. 1974). The

parental lines were grown in 5L pots at a mildew-free

greenhouse and were inoculated with fresh spores of

Bgt#66. In order to avoid the risk of cross-contami-

nation, transparent plastic cylinders were used to cover

plants prior to inoculation with Bgt spores. The pots

were then transferred to a controlled growth chamber

(15 �C) and were kept closed in those plastic

cylinders.

Statistical analysis

The JMP� ver. 10.0 statistical packages (SAS Insti-

tute, Cary, NC) were used for all statistical analyses.

The mean density of pustules (average of three leaf

segments) for each time point was transformed to

H(X ? 1) before analysis of variance. A factorial

model was employed for the analysis of variance

(ANOVA), with RILs and environment as random

effects. To assess the effect of RIL and environment

on the disease severity, ANOVA was implemented at

both 10DPI and 14DPI. Broad sense heritability

estimates (h2) were calculated for each trait across

the two environments using ANOVA-based variance

components:

Mol Breeding (2014) 34:1647–1658 1649

123

h2 ¼ r2G=ðr2

G þ r2G�E=EÞ

where r2G ¼ MSRIL � MSRIL�Eð Þ=E½ �; r2

G�E ¼MSRIL�E and E is the number of environments and

MS is the mean square.

QRL analysis

A genetic linkage map of 2,317 cM was previously

developed by genotyping the 152 RIL mapping

population with 197 simple sequence repeat (SSR)

and 493 Diversity Array Technology (DArT) markers

(Peleg et al. 2008). DArT markers presented in the

above map by clone ID numbers were renamed with

the prefix ‘‘wPt,’’ ‘‘rPt’’ or ‘‘tPt’’ (corresponding to

wheat, rye or triticale, respectively) followed by a

number. A skeleton map comprised of 307 markers,

scattered along the 14 chromosomes (chr.) of the

tetraploid wheat genome (an average of one marker

per 7.5 cM), was used for QRL analysis. The QRL

analysis was performed with the MultiQTL package

(http://www.multiqtl.com) using the general interval

mapping analysis for the RIL-selfing population as

described by Peleg et al. (2009a, b). QRL detection

was carried out with a structured multi-step scheme

embedded within the software (Korol et al. 2001).

First, the entire genome was screened for genetic

linkage using single-trait analysis (STA). Next, mul-

tiple interval mapping (MIM) was applied, which

incorporates interfering effects of other QRLs located

on a separate chromosome(s) into the model to reduce

the residual variation (Kao et al. 1999). MIM was

applied when more than one QRL was detected. The

hypotheses that a single locus or two-linked loci on the

considered chromosome could have an effect on one

or two quantitative traits were first tested by running

5,000 permutation tests (Churchill and Doerge 1994).

The hypothesis that one locus on the chromosome has

an effect on a given trait (H1) was compared with the

null hypothesis (H0) of no effect of the chromosome

on that trait. Once the genetic model was chosen,

5,000 bootstrap samples were run to estimate the

standard deviation of the main parameters: locus

effect, its chromosomal position, its LOD score, and

the proportion of explained variation (PEV). Finally,

to evaluate the genome-wide significance of estimates

obtained on a chromosome 9 trait basis, an approach

based on controlling the false discovery rate was used

to correct for multiple comparisons (Benjamini and

Hochberg 1995). The effect of an epistatic interaction

was examined by comparison of H0(e = 0), i.e.,

additive effects of the QRL, and H1(e = 0), i.e.,

assuming epistasis (Ronin et al. 1999).

Environmental specificity of QRL was determined

using two approaches. First, the two-environment

QRL model was compared against a sub-model

assuming equal effect of both environments, using

5,000 permutation tests to examine (Geno-

type 9 environment) interaction. To determine envi-

ronmental specificity, we used additional criteria

where the QRL must not only show significant

G 9 E interaction but must also show [0.05 differ-

ence between PEVs of the two environments. The

environment with the greater PEV value was declared

as the most influential. Second, when QRL was found

significant only for one environment but not for the

two-environment model, it was declared as an envi-

ronment-specific QRL.

Results

Screening of the parental lines with 42 Bgt isolates

This study is based on a cross between durum wheat

and wild emmer wheat. These two parental lines were

first tested for their response to a set of 42 Bgt isolates,

from the ‘‘Eshed-Dinoor mildew culture collection’’

collected from domesticated and wild wheat species

from different locations in Israel, and five isolates

from Switzerland collected from cultivated wheat

fields (Table S1, Ben-David et al. 2010). The wild

emmer wheat parent showed a resistance response to

29 of the Bgt isolates, while the durum wheat LDN

showed a resistance response to only a few isolates

(Ben-David et al. 2010). The wild parent (G18-16)

was highly resistant to the powdery mildew isolate

Bgt#15, whereas LDN was highly susceptible to this

isolate (IT scores 0 and 4, respectively). Therefore,

isolate Bgt#15 was previously used to map the

resistance loci harbored by the wild emmer wheat

parent, as a single dominant gene, designated PmG16

(Ben-David et al. 2010). Contrary to Bgt#15, Bgt#66 is

virulent on PmG16 and generated a unique quantita-

tive resistance reaction when tested on the parental

lines of the RIL population (Fig. 2S). The durum

wheat parent (LDN) showed slight powdery mildew

symptoms (IT = 1, designated R-), while the wild

1650 Mol Breeding (2014) 34:1647–1658

123

emmer parent (G18-16) showed a moderately suscep-

tible reaction (IT = 2–3, designated M). This reaction

of both parents was evident both at the seedling stage

(Zadoks index 12), as well as at the adult plant stage

(Zadoks index 57; Zadoks et al. 1974) (Fig. 1).

Quantitative phenotypic characterization

of inoculation with Bgt#66

Each Bgt isolate possesses specific phenotypic fea-

tures, such as mycelium growth rate, pustule produc-

tion rate and sporulation pattern, and therefore, one

needs to optimize the phenotypic measurements for

each isolate, prior to conducting large-scale experi-

ments. Based on the counting of pustule densities at

five time points, on five susceptible tetraploid lines

(Fig. 1S), we concluded that the most critical param-

eter in avoiding experimental error is the time of

measuring pustule densities after inoculation (DPI).

Early measurement of pustule densities could result in

false positives due to failing to distinguish between

established colonies and unsuccessful penetration sites

(manifested by hyper- sensitive-like responses), while

late counting may result in false negatives due to the

merging of mycelia of neighboring colonies. There-

fore, based on our preliminary assay, two time points,

10 and 14 DPI, were selected for further measurements

in order to optimize the accuracy of measurements of

pustule densities.

Table 1 presents the quantitative response, as

means of numbers of pustules per cm2 of leaf area,

for the two parental lines and the RIL population in

two environments at 10 and 14 DPI. G18-16 was

susceptible to Bgt isolate #66 with a mean range of

45.2–74.6 pustules per cm2 (Table 1), whereas LDN

was partially resistant with a mean range of 15.0–17.3

pustules per cm2 (Table 1). The RILs exhibited

transgressive segregation for quantitative resistance

to powdery mildew (range of 0–127.4 colonies per

cm2). The pustule densities measured in environment

II were prominently higher when compared to envi-

ronment I, while the two measurement points (10 and

14 DPI) within each environment were relatively

homogeneous. The susceptible wild emmer wheat

genotype 1–67 showed an average of 74.2 pustules per

cm2 across all replicates and was therefore used as a

G18-16LDN

a

b

c

d

Fig. 1 Phenotypic responses of the two parental lines, Langdon

(LDN) and G18-16, to inoculation with isolate Bgt#66. Leaf

segment and whole plant assays are presented: a, b phenotypic

response of LDN at adult plant stage and seedling stage,

respectively; c, d phenotypic response of G18-16 at adult plant

stage and seedling stage, respectively

Table 1 Mean values and data range of pustule densities at 10 and 14 days post-inoculation (DPI) of 152 F7 recombinant inbred

lines (LDN 9 G18-16) and the two parental lines under two environmental conditions (I and II)

DPI Environment I (pustules/cm2) Environment II (pustules/cm2)

RILs LDN G18-16 RILs LDN G18-16

Mean Range Mean Range

10 20 0–103.0 15 45.2 46.3 0.9–127.4 16.6 68.1

14 21.7 0–100.7 17.3 57.3 38.3 2.0–115.0 15.6 74.6

Mol Breeding (2014) 34:1647–1658 1651

123

positive control line in all experiments to monitor

infection conditions between inoculated boxes. The

average deposited spore density and the germination

rate values (number of spores with germ tubes/total

number of spores) for each box were measured in

order to ensure uniform inoculation density across the

assay. Analysis of variance of pustule densities carried

out on the segregating RIL population revealed highly

significant effects (P \ 0.001) of genotype and envi-

ronment at 10 and 14 DPI, significant interaction and

very high heritability (Table 2).

Characteristics of the detected QRLs

A whole-genome genetic linkage map of 2,317 cM

with an average of one marker per 7.5 cM was used for

dissection of the mechanism controlling the wheat

plant response to Bgt#66. A total of five significant

QRLs, scattered across 5 out of 14 chromosomes of the

tetraploid wheat, was detected under two environ-

ments at 10 and 14 DPI (Table 3; Fig. 2). In all QRLs

but one, the durum wheat allele (LDN) contributed to

improved powdery mildew resistance (lower density

of fungal colonies on the leaves). Only in one QRL

(5B), the favorable allele was contributed by the wild

parent (G18-16). Only the QRL on 7A exhibited a

significant G 9 E interaction. No significant two-

locus epistasis was detected between any pair of the

QRLs. Since the measurements of colony densities at

10 DPI provided, in most cases, a higher resolution, we

used the second measurement (14 DPI) as another

confirmation to the QRLs identified at 10 DPI.

A total of five significant QRLs conferring powdery

mildew resistance were identified at 10 DPI, with

LOD scores ranging between 3.4 and 16.3, and

explaining 1.3-26.6 % of the variance (Table 3).

Higher resistance was conferred by the LDN alleles

at four loci (1A, 2B, 6B and 7A) and by the G18-16

allele at one locus (5B). One QRL showed significant

G 9 E interaction (7A). Two QRLs (5B and 6B) were

detected only under environment II (Table 3). Three

significant QRLs conferring powdery mildew resis-

tance were detected at 14 DPI, with LOD scores

ranging between 3.9 and 19.8, explaining 0.3–26.4 %

of the variance (Table 3). These QRLs, detected at 14

DPI, provided further validation for the three major

QRLs detected at 10 DPI. The five significant QRLs,

detected at least in one of the two environments, at 10

or 14 DPI were designated as QPm#66-1A, QPm#-

662B, QPm#66-5B, QPm#-666B and QPm#-667A

(Fig. 2).

Discussion

Crosses between durum wheat cultivars are commonly

used in durum wheat genetic studies. However, the

narrow genetic variation possessed by these crosses

limits the ability to detect and enrich the durum

resistance gene reservoir with novel alleles. To

overcome this limitation, we have used a whole

genome analysis of a tetraploid wheat cross between

durum wheat and wild emmer wheat. Using this

approach, we were able to detect novel QRLs due to

the high variability of wild and domesticated wheat

germplasm. Moreover, the recent advances in the

development and use of high-density genetic and

genomic maps (Maccaferri et al. 2014a, b e.g., SNP

based) will greatly enhance the genetic detection

power and the chances to genetically dissect novel

plant resistance alleles and QRLs (e.g., Marone et al.

2013).

Based on multiple observations of responses to

different types of pathogens in various crop-plants,

quantitative disease resistance was suggested as more

durable than typical R-gene-mediated resistance

(reviewed by Parlevliet 2002; Marone et al. 2013).

The polygenic nature of QDR also affects the selection

pressure on the pathogen. Even if the pathogen

develops a mutation that enables it to overcome a

single QRL, the pathogen can gain only a marginal

Table 2 Analyses of variance for the effects of genotype and

environment on pustules’ density at 10 and 14 days post-

inoculation (DPI) in a segregating F7 RIL population

(LDN 9 G18-16)

Source of variation d.f. Mean square

10 DPI 14 DPI

Genotype (G) 136 2,652*** 1,959***

Environment (E) 1 135,825*** 55,784***

G 9 E 136 920*** 683***

Experimental error 548 393 301

Heritability (h2) 0.97 0.97

Heritability estimates (h2) of the RIL population are presented

for all traits

*** Significance at P \ 0.001

1652 Mol Breeding (2014) 34:1647–1658

123

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Mol Breeding (2014) 34:1647–1658 1653

123

advantage and therefore will have only a marginal

fitness advantage over other pathogen strains (Poland

et al. 2008). In the current study, we have used QRL

analysis to study plant–pathogen interactions and

discuss the results in light of previous QDR studies.

Bgt#66 was virulent on PmG16, but was avirulent

(IT = 0) on a set of 17 differential wheat lines that

carry Pm alleles (i.e., Pm1a, Pm1b, Pm2, Pm3a,

Pm3b, Pm3c, Pm3d, Pm4a, Pm4b, Pm5a, Pm5b, Pm6,

Pm7, Pm8, Pm9 and Pm17) (Ben-David 2011).

Therefore, isolate Bgt#66 allowed us to expose partial

resistance QRL alleles derived from the durum wheat

parental line. Five significant QRLs associated with

the phenotypic reactions to inoculation of tetraploid

wheat with Bgt#66 were identified and mapped.

Interestingly, most QRLs affecting improved resis-

tance to the Bgt#66 were conferred by the domesti-

cated durum wheat (LDN) alleles. These findings are

unique in the sense that, so far, only two Pm genes are

known to have originated from a durum wheat

background (Hsam and Zeller 2002; Huang et al.

2004; Zhu et al. 2004). A major QRL was detected on

chr. 1A (QPm#66-1A, LOD 16.3 and 19.8 for 10 and

14 DPI, respectively) explaining 26.6 and 26.4 % of

the variance in the two environments, at 10 and 14 DPI

(Table 3; Fig. 3). The implementation of the two

Fig. 2 Likelihood intervals for quantitative resistance loci

(QRLs) associated with resistance to powdery mildew isolate

Bgt#66 in recombinant inbred line (RIL) population of the cross

between Langdon and G18-16. Molecular markers are shown on

the left with map distances and QRLs on the right. The

approximate centromere position is indicated by black rectangle

1654 Mol Breeding (2014) 34:1647–1658

123

environments’ model in the QTL analysis has

improved the detection power of the analysis

(Fig. 3). Previously, two alleles of the Pm3 R-gene

were mapped to this region: Pm3 h, from Ethiopian

durum wheat (near marker Xgwm905; Huang et al.

2004), and Pm3 g from bread wheat (Sourdille et al.

1999, 2003). Recently, another QRL of field resistance

was mapped to the same location (Lu et al. 2012). A

comparison of the current QRL map with these genetic

maps shows that the Pm3 multi-allelic locus is located

on a distal genetic interval relative to QPm#66-1A.

QPm#66-2B was mapped to a genetic map interval

on chr. 2BL. Several adult plant resistance (APR) QRLs

were reported on chromosome 2BL (Bougot et al. 2006;

Liang et al. 2006; Tucker et al. 2007); however, they

were mapped to a more proximal location. QPm#66-7A

was mapped to the long arm of 7A. An APR QRL was

reported on the distal region of 7AL in proximity to the

Pm1 locus (Chantret et al. 2001), which is located

distally apart from QPm#66-7A.

Out of 42 Bgt isolates tested, LDN showed a

resistance response (IT = 0–2) to six isolates. It is not

clear whether the resistance response to these six

isolates is conferred by the five QRLs discovered in the

current study, in response to inoculation with Bgt#66, or

that other race-specific R-genes or QRL are involved.

Furthermore, it is not clear whether these QRLs

represent race-specific resistance conferred by LDN

in response to inoculation with Bgt#66, or that isolate

Bgt#66 represents a wider haplotype profile, which co-

exists with wild emmer populations in nature. In any

case, these newly discovered QRLs could serve as

sources for improved resistance. This is especially

relevant when taking into account the aspects of

pathogen populations’ genetic structure and dynamics,

and the risk of long-distance transfers of pathogens. In

addition, the era of climate change dictates, in general,

high unpredictability to any pathosystem. Therefore,

cautious selection, characterization and isolation are

needed for every resistance element in order to enrich

the stockpile of resistance genes and QRLs, even if, at

present, it is allegedly marginally effective.

Disease resistance may appear at different stages of

host development, varies with plant age or tissue

maturity, and it is highly related to plant–pathogen

interactions (Develey-Riviere and Galiana 2007). In

the current study, we have characterized the resistance

to powdery mildew at the seedling stage. Furthermore,

this resistance was also clearly evident in LDN, the

resistant parent of the mapping population at the adult

stages of development, as can be seen in Fig. 1.

Although a significant advancement was achieved in

recent years toward the understanding of the mecha-

nisms that underlie resistance, the association between

seedling and APR is not clearly defined. Moreover,

APR by itself is lacking an accurate and realistic

Fig. 3 Models for quantitative resistance loci analysis on chromosome 1A for two environments (red) and a single environment (blue

and green for environments I and II, respectively). (Color figure online)

Mol Breeding (2014) 34:1647–1658 1655

123

definition. Quantitative APR gene is considered to be

expressed when major APR resistance genes are

absent from the genetic background or when only a

defeated forms of the R-gene is evident (Wang et al.

2005). While it can be assumed that APR genes are

more durable, cases of race-specific APR loci were

also reported and therefore should be taken into

consideration when used by breeders (Li et al. 2014).

It has been suggested that some QRLs or APR

condition a weaker form of R-gene-mediated defense

although the possibility that other mechanisms could

provide isolate-specific resistance was not ruled out

(Parlevliet and Zadoks 1977). In light of this hypoth-

esis, phenotypic variance and durability can be

explained by a minor-gene-for-minor-gene interac-

tion, where virulence genes having a minor effect in

the pathogen correspond to resistance genes having a

minor effect in the host. Poland et al. (2008) assumed

that qualitative and quantitative disease resistance

might only be two ends of a continuum, with R-genes

and QRLs resembling the two extremes of the

spectrum. Although selection favors R-genes with

strong effects, pathogen evolution can erode the

effectiveness of R-genes, converting them into QRLs.

This phenomenon, known as ‘‘residual resistance,’’

has been observed in wheat powdery mildew (Nass

et al. 1981) and wheat stem rust (Brodny et al. 1986):

When a pathogen strain overcomes an R-gene, the

level of disease in the presence of the ‘‘defeated’’

R-gene is reduced relative to the level of disease in the

absence of the R allele. QTL analysis can help test the

hypothesis that QRLs are variants of R-genes that have

been (partially) overcome by their respective patho-

gens. Combining DNA markers and QTL mapping,

complex forms of disease resistance and their under-

lying genes are now far more accessible for investi-

gation (e.g., Marone et al. 2013). Positional cloning of

R-genes and QRLs is still a challenging task in wheat.

Only two partial resistance genes are cloned so far in

wheat, shedding some light on the mechanisms

involved (Yr36, Fu et al. 2009; Lr34, Krattinger

et al. 2009). Yr36 provides partial resistance to all

stripe rust races tested, while Lr34 confers resistance

to multiple pathogens. Hence, we assume that the

resistance mechanisms conferred by the QRLs

described in the current study are different from those

of Yr36 and Lr34. Further study is needed to reveal the

genetic and physiological differences between quali-

tative and quantitative disease resistance.

While the reaction to Bgt#66 expresses a partial

resistance response of the domesticated parent and

susceptible response of the wild parent, the response

to another isolate, Bgt#15, was susceptibility in

LDN and resistance in G18-16. This response

facilitated the mapping of an effective R-gene

(PmG16) on chr. 7A (Ben-David et al. 2010). Using

the same mapping population as in this study,

PmG16 was genetically and physically mapped to

the long arm of chromosome 7A, on wheat

chromosome deletion bin 7AL-16 0.86-1.00. Hence,

we have used the same genetic platform to identify

and map a new powdery mildew resistance gene

from wild emmer and to map new QRLs derived

from durum wheat. The high potential of wild

emmer wheat as a reservoir for new powdery

mildew resistance genes has been shown previously

(Ben-David et al. 2010; Xie et al. 2012). In contrast,

durum wheat is underrepresented in the current

published Pm catalog and could be an important

genetic source.

In conclusion, the genetic analysis of the pheno-

typic reactions of the RIL population at the seedling

stage to the two Bgt isolates describes two different

resistance mechanisms. The first is monogenic: a

wild emmer wheat allele in a single locus conferring

complete resistance to Bgt#15. The second is

polygenic: a set of durum wheat alleles, in five

independent QTLs, controls partial resistance to

Bgt#66 in the RIL population. Bgt belongs to a

group of pathogens, which holds the highest risk to

wheat crops due to their dual reproduction system

and a high degree of gene/genotype flow (McDonald

and Linde 2002). Thus, breeding efforts should be

focused on sources of quantitative resistance that

will need to be renewed regularly in order to be able

to stay ahead of the pathogen. The identified new

resistance alleles from durum wheat could contrib-

ute to wheat breeding for Bgt resistance by precise

exploitation of the available and well-studied LDN

genetic platform. The results of the current study

shed light on evolutionary mechanisms associated

with the development and distribution of powdery

mildew resistance genes in wheat.

Acknowledgments This study was supported by The Israel

Science Foundation Grant #205/08 and equipment Grants

1478/04 and #1719/08. The authors thank A. Fahoum and M.

Chatzav for their excellent technical assistance and to Dr.

T. Kis-Papo for scientific editing of the manuscript.

1656 Mol Breeding (2014) 34:1647–1658

123

Conflict of interest All authors of the manuscript have

declared no conflicts of interest.

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