THE PL ANT GENOME MARCH 2012 VOL. 5, NO.

1 1
ORI GI NAL RESEARCH
Association Mapping of Quantitative Trait Loci
in Spring Wheat Landraces Conferring Resistance
to Bacterial Leaf Streak and Spot Blotch
Tika B. Adhikari,* Suraj Gurung, Jana M. Hansen, Eric W. Jackson, and J. Michael Bonman
Abstract
Bacterial leaf streak (BLS), caused by Xanthomonas translucens
pv. undulosa (Smith et al.) Bragard et al., and spot blotch (SB),
caused by Cochliobolus sativus (S. Ito & Kurib.) Drechs. ex
Dastur, are two emerging diseases of wheat (Triticum aestivum
L.). To achieve sustainable disease management strategies and
reduce yield losses, identifying new genes that confer quantitative
resistance would benet resistance breeding efforts. The main
objective of this study was to use association mapping (AM) with
832 polymorphic Diversity Arrays Technology (DArT) markers to
identify genomic regions associated with resistance to BLS and
SB in 566 spring wheat landraces. From data analysis of this
diverse panel of wheat accessions, we discovered ve novel
genomic regions signicantly associated with resistance to BLS on
chromosomes 1A, 4A, 4B, 6B, and 7D. Similarly, four genomic
regions were found to be associated with resistance to SB on
chromosomes 1A, 3B, 7B, and 7D. A high degree of linkage
disequilibrium (LD) decayed over short genetic distance in the set
of wheat accessions studied, and some of these genomic regions
appear to be involved in multiple disease resistance (MDR).
These results suggest that the AM approach provides a platform
for discovery of resistance conditioned by multiple genes with
quantitative effects, which could be validated and deployed in
wheat breeding programs.
W
HEAT (Triticum aestivum L.) is a major crop
worldwide. Both abiotic and biotic stresses limit
successful wheat production. Among biotic stresses,
plant diseases have a negative impact on crop health
and are strongly infuenced by climate change (Sharma
et al., 2007). Bacterial leaf streak (BLS), caused by Xan-
thomonas translucens pv. undulosa (Bragard et al., 1997),
is emerging as a potential threat to wheat production in
the Upper Midwest of the United States in recent years
(Adhikari et al., 2011a) because most commercial wheat
varieties grown in the region appeared to be susceptible
to BLS (Adhikari et al., 2009). Bacterial leaf streak can
cause up to 40% yield loss (Forster and Schaad, 1988).
Te pathogen is seed borne (Forster and Schaad, 1985,
1988; Fourest et al., 1990) and can disperse long distance
via wheat germplasm exchange (Maraite et al., 2007).
Spot blotch (SB), caused by Cochliobolus sativus
(Ito & Kuribayashi) Drechsler ex Dastur [anamorph:
Bipolaris sorokiniana (Sacc.) Shoemaker], is another
emerging disease of wheat and barley (Hordeum vulgare
L.) (Knight et al., 2010; Duveiller et al., 2005; Zhong
Published in The Plant Genome 5:116. Published 7 Feb. 2012.
doi: 10.3835/plantgenome2011.12.0032
Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
An open-access publication
All rights reserved. No part of this periodical may be reproduced or
transmitted in any form or by any means, electronic or mechanical,
including photocopying, recording, or any information storage and
retrieval system, without permission in writing from the publisher.
Permission for printing and for reprinting the material contained herein
has been obtained by the publisher.
T.B. Adhikari, S. Gurung, and J.M. Hansen, Dep. of Plant Pathology,
North Dakota State Univ., NDSU Dep. 7660, P.O. Box 6050,
Fargo, ND 58108; E.W. Jackson and J.M. Bonman, USDA-ARS,
Small Grains and Potato Germplasm Research Unit, Aberdeen, ID
83210. Current address of S. Gurung: Dep. of Plant Pathology,
Univ. of California, Davis, c/o U.S. Agricultural Research Station,
1636 E. Alisal Street, Salinas, CA 93905. Received 28 July 2011.
*Corresponding author (tadhikari31@yahoo.com).
Abbreviations: AM, association mapping; AUDPC, area under
disease progress curve; BLS, bacterial leaf streak; DArT, Diversity
Arrays Technology; FDR, false discovery rate; GS, growth stage;
IR, infection response; LD, linkage disequilibrium; MAF, minor allele
frequency; MDR, multiple disease resistance; NDSU, North Dakota
State University; NSGC, National Small Grains Collection; PC,
principal component; PCA, principal component analysis; PCR,
polymerase chain reaction; pFDR, p value determined with the false
discovery rate; QTL, quantitative trait loci; RIL, recombinant inbred
line; SB, spot blotch; SNP, single nucleotide polymorphism; SSR,
simple sequence repeat.
Published March, 2012
2 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
and Stefenson, 2001) in North America (Ghazvini and
Tekauz, 2007, 2008; Windels et al., 1992) and in South
Asia (Duveiller and Sharma, 2009). Te disease has a
signifcant impact on wheat production (Joshi et al.,
2007; Siddique et al., 2006; Sharma et al., 2007) and
can cause up to 30% yield loss (Duveiller et al., 2005).
Chemical control to manage these diseases (Forster
and Schaad, 1985, 1988; Fourest et al., 1990; Sharma-
Poudyal et al., 2005; Duveiller et al., 2005) is neither
economical nor environmentally friendly. Terefore,
identifying genes that condition quantitative resistance
and developing durably resistant cultivars is the best
approach to manage both BLS and SB in wheat.
Evaluation of diferent plant populations at the
molecular levels and utilization of such information
in varietal improvement programs are imperative to
efectively use the genetic resources in wheat breeding
programs (Chao et al., 2007; Zhang et al., 2010).
Additionally, analysis of genetic diversity, population
structure, and linkage disequilibrium (LD) in populations
also provides valuable information for association
mapping (AM) and marker-assisted breeding (Chao
et al., 2007, 2010; Zhang et al., 2010). Wheat is a self-
pollinated crop and recombination frequencies of specifc
genetic regions appear to be low in selective breeding
populations undergoing a rapid rate of inbreeding with
selfng (Tester and Langridge, 2010; Zhang et al., 2010).
Compared to other cereal crops (e.g., barley, maize [Zea
mays L.], rice [Oryza sativa L.], and sorghum [Sorghum
bicolor (L.) Moench]), wheat has a large genome and
therefore it has the poorest marker coverage (Zhang et al.,
2010). Importantly, LD estimates in wheat also vary with
chromosomes across genome in a population (Breseghello
and Sorrells, 2006; Chao et al., 2007). Although linkage
mapping has been successfully used to map genes or loci
in biparental populations (e.g., recombinant inbred lines
[RILs], doubled haploid, or backcrosses), the AM approach
could be an ef cient strategy to detect quantitative trait
loci (QTL) particularly in genetically diverse germplasm or
wild relatives (Flint-Garcia et al., 2003). In this approach,
no prior information on markertrait associations is
necessary, and multiple loci can be readily identifed.
Several robust statistical tools and modeling methods such
as mixed linear models (Yu et al., 2006; Zhu et al., 2008),
Bayesian clustering (Pritchard et al., 2000), principal
component analysis (PCA) (Price et al., 2006), and Q+K
(terms that abbreviate gross structure based on given
number of principle components [Q] and fner structure
based on kinship [K]) mixed models (Yu et al., 2006) have
been developed and used to enhance our understanding of
complex traits in animal and plant genetic systems. Tese
techniques have been utilized to reduce the occurrence
of false positive markertrait associations (Kang et al.,
2008; Tommasini et al., 2007) and have been successfully
applied to detect QTL associated with disease resistances
in barley (Roy et al., 2010) and wheat (Ghavami et al., 2011;
Massman et al., 2011).
To detect potential QTL using the AM approach,
degree of marker polymorphism and genome coverage
are important aspects in wheat. Because of chromosome
specifcity, codominance, high reproducibility, and
high polymorphism, simple sequence repeat (SSR) or
microsatellite markers have been extensively used in wheat
(Huang et al., 2002; Roder et al., 1998). Recently, Diversity
Arrays Technology (DArT) (Canberra, Australia; http://
www.diversityarrays.com) markers were developed and
used in mapping diferent traits in wheat and other crops.
Diversity Arrays Technology markers are a cost-efective
high throughput technology that can detect all types of
DNA variations such as single nucleotide polymorphism
(SNP), copy number variation, and methylation in plant
genomes (Diversity Arrays Technology, 2011).
A few wheat cultivars (e.g., Turaco, Alondra,
Angostura, Mochis, and Pavon 76) identifed in
greenhouse inoculations and feld conditions were
useful sources of resistance to BLS (Duveiller et al.,
1993; Forster and Schaad, 1985). Reactions of wheat
germplasm to X. translucens pv. undulosa also were
found to be similar both in feld and in greenhouse
conditions (Akhtar and Aslam, 1986). Most recently,
several winter accessions resistant to multiple strains of
X. translucens pv. undulosa were identifed (Adhikari et
al., 2011a). However, molecular mapping of genes or loci
for resistance to BLS is not yet complete. Resistance to
SB in wheat was reported to be both quantitatively and
qualitatively inherited. Initially, four QTL conferring
resistance to the disease were mapped in the Chinese
wheat variety Yangmai 6 on chromosomes 2AS, 2BS,
5BL, and 6DS (Kumar et al., 2009). Using SSR markers
and area under disease progress curve (AUDPC) based
on disease severity (%) at adult plant stage, Kumar et
al. (2010) identifed four QTL for resistance to SB on
chromosomes 2AS, 2BS, 5BL and 7DS in the Ning 8201
Sonalika population (Kumar et al., 2010) and fve
other QTL on chromosomes 2BS, 2DS, 3BS, 7BS, and
7DS in another population Chirya 3 Sonalika.
Bacterial leaf streak and SB are caused by diferent
plant pathogen groups: prokaryotic and eukaryotic,
respectively. Pathogenesis and mode of nutrient uptake by
the two pathogens are distinct. Xanthomonas translucens
pv. undulosa enters through the stomata and grows
in the parenchyma (Cunfer, 1987). During infection,
elongated streaks limited by the veins are developed
and later yellow exudates are formed on the surface of
streaks (Cunfer, 1987). Infected glumes are discolored or
blackened (Broadfoot and Robertson, 1993; Smith et al.,
1919). For C. sativus, infection takes place when spores
land and appressoria and infection cushions form on the
host surface before penetration. Infection proceeds from
the epidermis to the cortex and endodermis, resulting
in breakdown of these tissues. Te fungus produces
phytotoxins that can kill host cells (Aggarwal et al., 2011).
Subsequently, this necrotrophic pathogen derives nutrients
from dead host tissues, which enables pathogen growth
and disease development. Terefore, several aspects of
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 3
pathogenesis are unique to each disease and could require
distinct host defense mechanisms, signal transduction
pathways, transcription factors, and genes that confer
disease resistance. Available evidence suggested that
multiple disease resistance (MDR) genes exist in plants
(Wisser et al., 2005, 2011) and that a locus conditions
quantitative resistance to multiple diseases (Wisser et al.,
2011). Positional cloning of a putative ABC transporter
gene in wheat (Krattinger et al., 2009), silencing of a
germin-like protein gene family in rice (Manosalva et al.,
2009), and multivariate analytical approach for structured
AM in maize (Wisser et al., 2011) have verifed genes
involved in MDR.
Both BLS and SB are emerging diseases of wheat
in the Upper Midwest of the United States possibly due
to climate change. Despite their economic importance,
quantitative resistance to these diseases is poorly
understood. Wheat landrace accessions from the
USDA National Small Grains Collection (NSGC) are a
rich source of disease resistance genes (Bonman et al.,
2006, 2007). Our previous studies demonstrated that
the combined DArT markers and AM strategy were
efective for identifying QTL associated with resistance
to Phaeosphaeria nodorum (Adhikari et al., 2011b)
and Pyrenophora tritici-repentis in wheat (Gurung et
al., 2011). Tese fndings have prompted us to test the
hypothesis that there also exists naturally occurring
allelic variation for resistance to BLS and SB in these
wheat landraces representing diverse origin. Ultimately,
QTL associated with resistances that map to new loci
within the wheat genome would likely represent new
resistance sources and would be benefcial in eforts
to breed resistance to these two diseases either for
cultivar release or use as parents in breeding programs.
Association mapping should be a useful tool to identify
such loci within the collection materials. Te main
objective of this study was to discover novel loci for
resistance to BLS and SB using AM in a set of 566 spring
wheat landraces from the NSGC. Once it is known that
novel resistance is present within the set of accessions
evaluated, further work will be needed to validate the
resistance and transfer it to adapted genetic backgrounds.
MATERIALS AND METHODS
Wheat Accessions
In total, 566 wheat accessions with spring habit from
the NSGC used in previous association analysis for P.
nodorum (Adhikari et al., 2011b) and P. tritici-repentis
(Gurung et al., 2011) were analyzed in this study. Tese
accessions were classifed as landraces and represent
diverse geographic origin. To avoid any efects of con-
founding factors (e.g., growth stages, dual infection of
leaf spot pathogens, environmental factors), all experi-
ments were conducted in controlled environments at
North Dakota State University (NDSU), Fargo, ND, dur-
ing the fall of 2009 and the winter of 2010.
Phenotypic Evaluation
Planting Wheat Accessions and Experimental Designs
For BLS, three seeds of each accession were planted in each
of three cones (Stuewe and Sons, Inc., Corvallis, OR). To
the soil in each container, 2.5 g of multicote slow release
commercial fertilizer (141416 NPK; Sungro Horticul-
ture Distribution Inc.) was applied at the time of planting.
Wheat breeding line ND495 (Adhikari et al., 2009) and
winter wheat cultivar Magnum (Milus et al., 1996) served
as susceptible and resistant controls, respectively. Wheat
accessions were arranged in a randomized complete block
design (RCBD) with three replications. Each treatment
consisted of three plants per replication. Each cone was
considered as an experimental unit, and the fag leaf of
each plant was regarded as a sampling unit. Greenhouse
temperatures were maintained at a temperature of 24 to
28C with a 16 h photoperiod using 400 W Lucalox LU400
sodium vapor lamps (General Electric Co.) to supplement
natural light until disease scoring.
For SB evaluation, two experiments were conducted
in growth chambers at the same time using procedures as
described previously (Gurung et al., 2011). Planting and
experimental design were the same as described above.
Wheat cultivars Sonalika (Sharma et al., 2007) and
Chirya 7 (Kumar et al., 2010) served as susceptible and
resistant controls, respectively. Each cone was considered
an experimental unit and each single plant in a cone
was regarded as a sampling unit. Wheat accessions were
arranged in a RCBD with three replications. In each
experiment, the wheat accessions were fxed efects and
the replications were random efects.
Inoculum and Inoculation
Te highly virulent strain BLS-CS42 (formerly BLSW16) of
X. translucens pv. undulosa from Casselton, ND, was used
in this study. Inoculum preparation and inoculation proce-
dures were similar to those described previously (Adhikari
et al., 2011a). Briefy, the bacterial strain was cultured on
peptone sucrose agar (Ou, 1985), suspended in water, and
the inoculum density was adjusted to approximately 1 10
7

colony forming units ml
1
using a spectrophotometer. Ten
to 15 L of inoculum was infltrated into a fully expanded
fag leaf using a needleless disposable syringe, which infl-
trates without wounding (Milus and Mirlohi, 1994). Te
infltrated areas were marked by a permanent marker and
the plants were placed in trays of water and lef in the green-
house. Greenhouse temperatures were maintained at 26 to
28C with 16 h of supplemental light provided by 400 W
Lucalox LU400 sodium vapor lamps (General Electric Co.)
until disease assessment.
An isolate CS45 of C. sativus originally collected from
Bhairahawa, Nepal, was imported (USDA-APHIS permit
no. P526P-09-02536) to NDSU, Fargo, ND. Inoculum
preparation and inoculation procedures were the same
as described previously (Mahto et al., 2010, 2011). Briefy,
a mycelium plug was placed on V8 potato dextrose
agar consisting of 150 mL of V8 juice (Campbell Soup
4 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
Company), 10 g of potato dextrose (Becton, Dickinson and
Company), 10 g of Difco agar, 3 g of calcium carbonate,
and 850 mL of sterile distilled water in 10-cm petri dishes.
Te plates were incubated for 8 to 10 d at 24C and conidia
were suspended in water and the inoculum was adjusted to
3.5 10
3
conidia ml
1
using a hemacytometer. Two-week-
old seedlings were spray inoculated until run-of with the
suspension and seedlings were immediately incubated
in a mist chamber for 24 h at 100% relative humidity at
24C. Finally, plants were moved to a growth chamber and
programmed for a 23/19C diurnal cycle (day/night) with a
16-h photoperiod.
Phenotypic Data Analysis
In the previous study (Adhikari et al., 2011b), we used
stringent disease assessment methods such as high bacte-
rial inoculum concentration (10
7
colony forming unit ml
1
)
and disease rating scale (06 score) under controlled envi-
ronments. We found that median disease scores of 2.0
(i.e., less than 10% water-soaking and chlorosis symptoms
on the fag leaf) was a reliable criterion to classify resistant
and susceptible wheat accessions. Terefore, the same
approach was used here. For BLS, disease reactions within
infltrated areas (0.2 to 0.4 cm
2
) on the fag leaves were
assessed between 7 and 9 d afer infltration using a 0 to 6
rating scale (Milus and Chalkly, 1994), in which 0 equals
no visible symptoms, 1 equals chlorosis without water-
soaked lesions, 2 equals water soaking less than 10%, 3
equals water soaking 10 to 30%, 4 equals water soaking 31
to 70%, 5 equals water soaking 71 to 100%, and 6 equals
water soaking extending beyond the infltrated area.
Median disease scores of 0 to 2 were considered resistant
(Tillman et al., 1996) whereas median disease scores of >2
to 6 were regarded susceptible.
Although AUDPC based on disease severity (%) has
been used to assess resistance to SB at adult plant stage
(Kumar et al., 2010), our goal was to evaluate resistance
to SB at seedling stage in a controlled environment. For
SB, we used the rating scales recommended by Fetch and
Stefenson (1999). In this method, infection responses (IRs)
on wheat seedlings infected with C. sativus were estimated
based on the type (presence of necrosis and chlorosis)
and relative size of lesions developed on the second leaves
(Fetch and Stefenson, 1999). For phenotypic analysis, two
interactions were assessed 9 d afer inoculation. Minute
to small necrotic lesions (with no or very slight difuse
marginal chlorosis) characteristic of IRs 1, 2, 3, and 4 were
considered resistant while medium-sized to large necrotic
lesions with a distinct but restricted chlorotic margin or
even expanding difuse chlorosis characteristic of IRs 5, 6,
7, 8, and 9 were considered susceptible to highly susceptible
(Fetch and Stefenson, 1999). Nonparametric analysis
(Shah and Madden 2004) was performed to estimate
median disease scores and relative treatment efects (p
ij
)
for BLS and SB using Proc Mixed of SAS (SAS Institute,
2010). Additionally, Bartletts
2
were calculated to test the
hypothesis of homogeneity of variances as described in
previous work (Gurung et al., 2011).
DNA Extraction and Genotyping
Te same DNA used previously for P. nodorum
(Adhikari et al., 2011b) and P. tritici-repentis (Gurung
et al., 2011) was tested for AM of resistance to X. trans-
lucens pv. undulosa and C. sativus in this study. Deoxy-
ribonucleic acid samples were shipped to Triticarte Pty.
Ltd. for DArT genotyping. In total, 2500 DArT markers
developed from a PstI and BstNI combination were used
to identify QTL in spring wheat landraces as described
previously (Akbari et al., 2006). A DArT consensus map
was developed using a set of 80 wheat populations (RILs,
doubled haploid, and F
2
) (Akbari et al., 2006; A. Kilian,
personal communication, 2011).
Association Mapping Analysis
All computer sofware and data analyses used in this
study were similar as described in previous works
(Adhikari et al., 2011b; Gurung et al., 2011). Briefy, the
JMP Genomics sofware (SAS Institute, 2010) and the
TASSEL program, version 2.1 (Bradbury et al., 2007)
were used to analyze the marker properties, LD, princi-
pal component (PC) matrix, hierarchical clustering, and
Q+K mixed model. From the original DArT marker data
set, loci with minor allele frequencies (MAFs) < 0.06 or
with more than 0.13 missing genotypes were removed
from further analysis as were markers lacking map posi-
tions. In total, the 832 polymorphic DArT markers were
detected across the wheat accessions. Among these, the
625 had specifc chromosomal locations (Dr. A. Kilian,
Diversity Arrays Technologies, Canberra, Australia,
personal communication, 2011). Te MAFs and the
proportion of missing genotypes were evaluated for 832
polymorphic markers. Linkage disequilibrium statistics
(R
2
) were calculated using the TASSEL program. For
each chromosome, LD values were calculated separately
and later were combined across three wheat genomes A,
B, and D as described previously (Emebiri et al., 2010).
Te 95 percentile of unlinked LD estimates was used
to derive the critical value of R
2
(Breseghello and Sor-
rells, 2006). Te intergenic R
2
values were plotted against
genetic distance and a curve was ftted to the plot in SAS
9.2 (SAS Institute, 2010). Te intrachromosomal R
2
val-
ues against the genetic distance were plotted, and later a
distance at which the critical R
2
was determined by plot-
ting a LOESS curve (Neumann et al., 2011).
All pairwise marker-to-marker (n = 625) correlation
coef cients were calculated and used to perform (Q)
PCA and develop a pairwise allele sharing similarity
matrix (K). To visualize structure within the 566 wheat
accessions, Wards clustering method, which employs an
agglomerative clustering algorithm (Ward, 1963), was
used. Data from the appropriate number of principle
components (3) and the allele sharing similarity matrix
account for Q and K, respectively, in a linear model
to associate numeric DArT genotypes with ordinal
phenotypes using residual denominator degrees of
freedom. Te residual pseudo-likelihood estimation
(RSPL) method in PROC GLIMMIX was used with no
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 5
constraint of the K matrix covariance parameter because
the discrete distribution of the ordinal trait was not
normal. Since dominance or additive efects were not
assumed, association tests were performed by treating
genotypes as categorical variables in ANOVA (dominance
model) and as quantitative variables in regression (additive
model) analyses. Te negative Log
10
(NegLog
10
) conversion
was used on all calculated p-values. All of the tests were
performed with and without the false discovery rate (FDR)
multiple testing correction. Based on the F statistic and
numerator and denominator df value, the phenotypic
variation explained by a particular marker (R
2
) was
calculated (Edwards et al., 2008).
RESULTS
Phenotypic Analysis
Artifcial infections on wheat accessions under controlled
conditions for both diseases were highly reproducible and
consistent across replications or experiments, and also
were comparable to resistant and susceptible checks. For
BLS, wheat accessions were highly signifcant (df = 565,
F-value 11.92, and p 0.001) and no signifcant interaction
(p 0.0001) between wheat accessions and replications
was observed (data not shown). Since phenotypic data
were homogeneous (Bartletts
2
= 11.13 and p = 0.112),
BLS data were combined and overall means were used for
AM to identify putative genomic regions associated with.
For SB, no interaction was found between two experi-
ments (Bartletts
2
= 4.28 and p 0.05; data not shown).
Terefore, data from both experiments were pooled, and
overall means were used in AM study.
Nearly 31.9 and 25.0% of the accessions were
resistant to BLS and SB, respectively (Fig. 1A). Median
disease scores of resistant and susceptible checks to
BLS were 1 and 4, respectively (Supplemental Table S1).
Among the BLS-resistant accessions, 43 accessions had
disease scores between 0 and 1 while 138 accessions
had disease scores between 1.1 and 2 (Fig. 1A). Median
disease scores of resistant and susceptible checks to SB
were 3, and 8, respectively (Supplemental Table S2). Of
the SB-resistant accessions identifed, four of the resistant
accessions had mean disease score less than 3 and the
remaining resistant accessions had mean disease scores
between 3.1 and 4 (Fig. 1B).
Phenotypic Analysis Based on Geographic Origin
Signifcant diferences were observed for the proportion of
resistant accessions when classifed by geographic origin
(Table 1). Te percent of BLS resistant accessions from
Africa was signifcantly higher than for those originating
from Asia. In contrast, SB resistance was more frequent
Figure 1. Disease reactions of 566 spring wheat landraces to Xanthomonas translucens pv. undulosa, the causal agent of bacterial leaf
streak (BLS) (A), and Cochliobolus sativus, which causes spot blotch (SB) (B). For BLS, disease reactions on inltrated areas (0.2 to 0.4
cm
2
) on ag leaves were assessed 7 to 9 d after inltration using a 0 to 6 rating scale (Milus and Chalkly, 1994) in which 0 equals
no visible symptoms, 1 equals chlorosis without water-soaked lesions, 2 equals water soaking less than 10%, 3 equals water soaking
10 to 30%, 4 equals water soaking 31 to 70%, 5 equals water soaking 71 to 100%, and 6 equals water soaking extending beyond
the inltrated area. Disease scores of the nine inltrated areas on ag leaves (one site on each ag leaf) were analyzed. Median
disease scores of 0 to 2 were considered resistant (Adhikari et al., 2011a; Tillman et al., 1996) whereas median disease scores of >2
to 6 were regarded as susceptible. Wheat breeding line ND 495 (Adhikari et al., 2009) and winter wheat cultivar Magnum (Milus et
al., 1996) served as susceptible and resistant controls, respectively. For SB, infection responses (IRs) on wheat seedlings infected with
Cochliobolus sativus were estimated according to the type (presence of necrosis and chlorosis) and relative size of lesions developed
on the second leaves (Fetch and Steffenson, 1999). Minute to small necrotic lesions (with no or very slight diffuse marginal chlorosis)
characteristic of IRs 1, 2, 3, and 4 were considered resistant while the medium-sized to large necrotic lesions with a distinct but
restricted chlorotic margin or even expanding diffuse chlorosis characteristic of IRs 5, 6, 7, 8, and 9 were considered susceptible to
highly susceptible (Fetch and Steffenson, 1999) 9 d after inoculation. Wheat cultivars Sonalika (Sharma et al., 2007) and Chirya 7
(Kumar et al., 2010) were used as susceptible and resistant controls, respectively.
6 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
in accessions from the Americas versus Asia and Africa.
Some countries bordering the Mediterranean showed a
high percentage of BLS-resistant accessions. For example,
collectively 58.7% of the accessions (44 of 75) from Tunisia
(9 of 9), Libya (1 of 1), Syria (4 of 5), Macedonia (3 of 4),
Egypt (3 of 5), Greece (7 of 12), Israel (2 of 4), and Turkey
(12 of 29) were resistant (Supplemental Table S1). Among
the accessions from the Americas, all entries from Peru (n
= 6), Guatemala (n = 3), and Honduras (n = 2) were resis-
tant to SB (Supplemental Table S2).
Marker Statistics and Linkage Disequilibrium
Among 832 polymorphic DArT markers, 85 with MAFs
of <0.06 and 122 markers without map positions were
removed. In total, 625 markers having MAFs >0.06 and
less than 0.13 missing genotypic data were used in the
analysis. Te majority of DArT markers were distributed
across wheat A and B genomes (39.7 and 38.1%, respec-
tively) while the D genome had the fewest (22.2%). Te
highest number of DArT markers was distributed on chro-
mosome 6A followed by chromosomes 1A, 3B, and 7D
(Fig. 2). Te average number of DArT markers per chro-
mosome was 54.5; however, the distribution was uneven
with only one marker present on chromosomes 5A and
5D and no markers present on chromosome 4D (Fig. 2).
A critical value for signifcance that was also considered
as an upper limit was fxed at R
2
= 0.049. Te genomewide
R
2
estimate was found to decline rapidly to 0.2 within 5
cM of genetic distance during the LD analysis (data not
shown). Specifcally, LD decayed rapidly (2 cM) on 9 of
the 17 chromosomes having more than one unique posi-
tion while chromosomes 1D, 6B, and 6D had the highest
LD when more than 12 unique DArT positions were used.
Only three unique DArT positions (11, 37.5, and 151.8
cM) were retained for the analysis on chromosome 3D
although it had the highest extent of LD (18 cM).
Population Structure and Relationship Matrix
Tree PCs accounted for 59.7% of the genetic variation in
566 spring wheat landraces. Te frst PC explained 40.5%
while the second and third PC explained 10.5 and 8.7%
of the variation, respectively (Fig. 3A). Structure shown
by the allele sharing similarity matrix was in line with
Table 1. Disease reactions of 566 spring wheat
landraces of diverse geographic origin to bacterial leaf
streak (BLS) and spot blotch (SB) in the greenhouse in
North Dakota.
Region n
No. of resistant
to BLS

Percent
resistant

No. of resistant
for SB
Percent
resistant
The Americas 38 12 31.6 ab 22 57.9 b
Europe 63 27 42.9 ab 17 27.0 a
Asia 390 105 26.9 a 82 21.0 a
Africa 72 36 50.7 b 19 26.4 a
Total

566 180 140

For BLS, disease reactions on inltrated areas (0.2 to 0.4 cm

2
) on ag leaves were assessed 7 to
9 d after inltration using a 0 to 6 rating scale (Milus and Chalkly, 1994). Disease scores of 0 to 2
(or less than 10% water soaking within the inltrated areas) were considered resistant (Tillman et
al., 1996) whereas disease scores of >2 to 6 (or greater than 10% water soaking) were regarded
susceptible. For SB, second leaves were rated 9 d postinoculation using 1 to 9 disease rating scale
(Fetch and Steffenson, 1999), in which disease scores 4 were considered resistant and those >4
were considered susceptible.

Percentages followed by the same letter not signicantly different by nonoverlap of the 95%
binomial condence interval.

Two accessions from the former Soviet Union and one accession of unknown origin included in total.
Figure 2. Distribution of 625 Diversity Arrays Technology (DArT) markers across wheat chromosomes. Chromosomal locations and
positions of DArT markers were based on information from Diversity Arrays Technology PTL (Canberra, Australia).
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 7
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8 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
the three PCs during PCA (Fig. 3A; Supplemental Table
S3) and the three main groups corresponded roughly
to geographic origin with most of the Eastern Africa,
Eastern Asia, South America, and Southern Europe
accessions grouping in one cluster, most of the Western
Europe accessions in a second cluster, and most of the
South-Central Asia accessions in a third cluster.
Cluster analysis (Ward, 1963) using the allele sharing
similarity matrix and the three PCs (data not shown)
showed 12 subpopulations or clusters in the entire
population (Fig. 3B; Supplemental Table S3). Cluster 12
contained the greatest number of accessions (n = 110)
followed by cluster 1 (n = 68) and cluster 5 (n = 64),
respectively. Clusters 11 (n = 16) and cluster 8 (n = 20) had
the fewest accessions while the number of accessions in
the remaining clusters ranged from 26 to 63 (Supplemental
Table S3). Te results based on pairwise allele sharing, as
displayed in the heat map (Fig. 3B), showed a large portion
of accessions in clusters 9, 10, and 11 shared similar alleles.
Accessions in these clusters also shared about 60% of
alleles with those in clusters 5, 6, and 7 (Fig. 3B).
Association Analysis of QTL Associated
with Resistance to Bacterial Leaf Streak
Putative genomic regions conferring resistance to BLS
were detected on chromosomes 1A, 4A, 4B, 6B, and 7D
afer correcting for multiple testing using the p value
determined with the false discovery rate (pFDR) crite-
rion. One distinct genomic region on chromosome 1A
spanning the interval of 60.7 to 75.2 cM was found signif-
cantly associated with three DArT markers (Table 2; Fig.
4A). Among the DArT markers, two markers (wPt-665259
and wPt-671483) were co-segregating and phenotypic
variations ranged from 1.4 to 1.6% (Table 2). Te other
four DArT markers (wPt-2780, wPt-8292, wPt-663764, and
wPt-5674) on chromosomes 4A, 4B, 6B, and 7D, respec-
tively, also were found signifcantly (p < 0.01 or p < 0.05)
associated with resistance and explained 1.5 to 2.6% of the
phenotypic variation (Table 2; Fig. 4C through 4G).
Association Analysis of QTL Associated
with Resistance to Spot Blotch
Putative genomic regions conferring resistance to SB were
detected on chromosomes 1A, 3B, 7B, and 7D afer cor-
recting for multiple testing using the pFDR criterion. One
candidate genomic region on chromosome 1A at 58 cM
was found signifcantly (p < 0.01 and p < 0.001) associated
with two co-segregating DArT markers, wPt-730148 and
wPt-668214 (Fig. 4A), and phenotypic variations ranged
from 1.7 to 2.1% (Table 2). In addition, markers wPt-1159
and wPt-5769 on chromosome 3B and wPt-2838 and wPt-
664459 on chromosome 7B and 7D were signifcantly
associated with resistance to SB (Table 2; Fig. 4F and 4G).
DISCUSSION
Although traditional breeding strategies have signifcantly
contributed to crop improvement, there has been slow
progress in developing wheat cultivars resistant to BLS
and SB (Sharma et al., 2007; Duveiller et al., 1993) possibly
due to resistance being complex and polygenic (Duveiller,
1990; Duveiller et al., 1993). Other confounding problems
for assessing resistance to BLS and SB are the infuence of
environmental factors, relying on natural infection, and
the efects of multiple leaf spot pathogen infections on the
expression of resistance under feld conditions (Duveiller
et al., 2005; Sharma and Duveiller, 2007). In this study,
the combination of highly reproducible phenotyping data
obtained from artifcial infections under the controlled
conditions, DArT genotyping technology, and the AM
approach were applied to identify QTL responsible for BLS
and SB, the two emerging bacterial and fungal diseases of
wheat, respectively. Considering population structure and
FDR, fve and four genomic regions were found signif-
cantly associated with resistance to BLS and SB, respec-
tively. Linkage disequilibrium decayed over short distance
in the panel of wheat accessions analyzed and some of the
novel genomic regions found to be involved in resistance
to the BLS and SB isolate used.
Sources of resistance to X. translucens pv. undulosa
were previously identifed in wheat cultivars Turaco,
Alondra, Angostura, Mochis, and Pavon 76 (Duveiller et
al., 1993; Forster and Schaad, 1985). However, no further
eforts were made to develop mapping populations from
these resistant cultivars and to map the resistance QTL
or genes on wheat chromosomes. Te QTL responsible
for resistance to BLS identifed in this study are novel
and the frst to be associated with specifc wheat
Table 2. Bacterial leaf streak (BLS) and spot blotch (SB)
phenotypic variation explained by signicant Diversity
Array Technology (DArT) loci based on association
analysis in a set spring landraces from the USDA
National Small Grains Collection.
Disease DArT allele Chromosome

Position

p-value

R
2
(%)

BLS wPt-4886 1A 60.7 2.29* 1.4

wPt-665259 75.2 2.51** 1.6
wPt-671483 75.2 2.56** 1.6
wPt-2780 4A 90.6 2.37* 1.5
wPt-8292 4B 110.8 2.39* 1.5
wPt-663764 6B 31.7 2.59*** 1.6
wPt-5674 7D 174.2 3.82*** 2.6
SB wPt-730148 1A 58.0 3.08*** 2.1
wPt-668214 58.0 2.51*** 1.7
wPt-1159 3B 53.2 2.40*** 1.6
wPt-5769 86.1 3.12*** 2.2
wPt-2838 7B 223.5 3.21*** 2.2
wPt-664459 7D 175.1 2.37* 1.5
*Signicant at the 0.05 probability level.
**Signicant at the 0.01 probability level.
***Signicant at the 0.001 probability level.

Chromosomal position of the signicantly associated DArT markers based on WheatDArTmaps version
(Diversity Arrays Technology, 2011).

The p-value adjusted after multiple tests (experimentwise p value). The signicance was assessed
using 10,000 permutations (Roy et al., 2010).

Phenotypic variation explained by the markers and not including the other items in the model.
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 9
chromosomes based on the consensus map assignment
of DArT alleles. Te potential sources of resistance to
multiple diseases also were identifed and mapped in
wheat landraces using DArT markers and AM approach
(Adhikari et al., 2011a; Gurung et al., 2011).
Kumar et al. (2009) identifed four QTL (Qsb.bhu-2A,
Qsb.bhu-2B, Qsb.bhu-5B, and Qsb.bhu-6D) based on
the reaction of Yangmai 6 Sonalika RILs to the most
aggressive C. sativus isolate (isolate no. International
Collection of Microorganisms from Plants [ICMP]
13584). Tese QTL were associated with resistance
to SB and explained by 63% of phenotypic variation.
Kumar et al. (2010) used the same isolate of C. sativus
from New Zealand and recorded disease severity (%)
in the feld at three diferent growth stages (GSs) such
as GS 63 (beginning of anthesis to half complete), GS
69 (anthesis complete), and GS 77 (late milk). Disease
severity values were used to estimate AUDPC in the Ning
8201 Sonalika and Chirya 3 Sonalika populations
and eight QTL (Qsb.bhu-2A, Qsb.bhu-2B, Qsb.bhu-2D,
Qsb.bhu-3B, Qsb.bhu-5B, Qsb.bhu-6D, Qsb.bhu-7B, and
Qsb.bhu-7D) were mapped using SSR markers (Kumar
et al., 2009, 2010). In the present study, we used highly
aggressive isolate (CS45) of C. sativus from Nepal and
DArT markers to identify potential genetic regions
associated with seedling resistance to SB. Terefore, it
is not feasible to directly compare the locations of these
QTL with the previous studies (Kumar et al., 2009, 2010)
due to diferent fungal isolates, crop growth stages, and
molecular marker systems used. Although the isolate was
used in this study was diferent from the previous studies
(Kumar et al., 2009, 2010), the two QTL (1A and 7B)
responsible for seedling resistance to SB were identifed
here and appeared to be novel. Whether or not these
two QTL for resistance to SB at seedling stage are also
efective to SB at adult plant stage needs to be examined.
Figure 4. Association mapping of resistance to bacterial leaf streak (BLS), caused by Xanthomonas translucens pv. undulosa, and spot
blotch (SB), caused by Cochliobolus sativus. Consensus maps are based on information from Diversity Arrays Technology (DArT) PTL
(Canberra, Australia). On the right, values are genetic distance in centimorgans (cM). On the left, DArT markers (bold) are signicantly
associated with resistance to BLS (blue color) and SB (red color) on the short and long arms of the chromosomes. In the current gures,
the genetic linkage maps align directly with the p-value plot. In negative log10 p plots (right), the dotted line indicates the signicance
threshold without false discovery rate correction and values are marked with * indicating signicant markers. Level of signicance of
p-value for each marker is given in Table 1.
10 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
Te AM approach relies on LD between pairs of
loci in natural populations (Buckler et al., 2001). On an
average, LD decay within 5 cM was observed for the A,
B, and D genome in this study. Linkage disequilibrium
decay ranged from 10 to 40 cM in previous studies (Chao
et al., 2007; Crossa et al., 2007; Emebiri et al., 2010). Te
obtained maps were based on genotyping with DArT
markers and do not allow us to correlate the genomic
regions found to be involved in resistance in this study
with other studies using polymerase chain reaction
(PCR)-based SSR or other PCR-based and/or restriction
fragment length polymorphism (RFLP) markers. In
addition, diferent population sizes, wheat genotypes,
wheat classes, and marker systems were used for
estimating LD decay in this study and in previous studies
(Chao et al., 2007; Crossa et al., 2007; Emebiri et al.,
2010). Terefore, the data are of limited value for a better
understanding of the molecular basis of wheat disease
resistance. Despite these limitations, we found signifcant
LD extending over 5 cM, indicating the window of
opportunity for identifying markertrait association in
this study was suf cient based on marker coverage across
Figure 4. Continued.
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 11
the genome with the exception of 3D, 4D, 4D, 5A, and
5D (Fig. 2). However, SNP marker-based genotyping is
available now in wheat and the fndings presented here
can later be extended using new markers.
In this study, QTL associated with BLS and SB
resistances had small phenotypic efects (R
2
) ranging
from 1.4 to 2.6% and from 1.5 to 2.2%, respectively. More
recently, we also detected small phenotypic variation
efects for P. nodorum (Adhikari et al., 2011a) and P.
tritici-repentis (Gurung et al., 2011) in wheat. Similarly,
low phenotypic variations in multiple traits have been
reported previously. For examples, QTL associated with
Fusarium head blight resistance had small phenotypic
efect (1 to 3%) due to resistance being complex and
polygenic inheritance (Massman et al., 2011). Shi et
al. (2011) identifed QTL signifcantly associated with
resistance to bacterial blight in common bean (Phaseolus
vulgaris L.) accounting for 0.04 to 4.9% of the phenotypic
variation. Association mapping of quantitative resistance
to Southern leaf blight in maize also identifed QTL with
small, additive efects (Kump et al., 2011). Additionally,
AM of resistance to SB in a barley population identifed
QTL with a small phenotypic variation ranging from 2.3
to 3.9% (Roy et al., 2010). Our fndings are in agreement
with the previous reports that resistance to these two
emerging diseases is complex and are likely conditioned
by multiple genes with quantitative efects (Duveiller
et al., 1993; Kumar et al., 2009, 2010). Te occurrence
of resistance also varied with geographic origin and
difered based on DArT marker haplotype cluster.
Terefore, resistant accessions of diferent geographic
origin or haplotype clusters could carry diferent
resistance genes. Landraces ofen have poor agronomic
characteristics and prebreeding will likely necessary to
transfer resistance into adapted wheat elite lines for use
by breedingprograms.
Using a set of interrelated standard genetic panels in
maize and rice, evidence for the presence clusters of genes
or QTL conferring MDR has been well demonstrated
(Ramalingam et al., 2003; Wisser et al., 2005, 2011).
Tese fndings provide convergent evidence underlying
specifc chromosomal segments and genes that control of
broad-spectrum resistance. Candidate genes involved in
MDR are being identifed and verifed through a range
of functional genomics criteria, including microarray
experiments identifying diferentially expressed genes,
clusters of genes with correlated expression, through
mutant analysis and gene silencing in rice (Fukuoka
et al., 2009; Manosalva et al., 2009), and multivariate
test statistic using the AM approach and resequencing
analysis of a candidate gene in involved in MDR (Wisser
et al., 2011). In wheat, a few DArT markers coincided with
Figure 4. Continued.
12 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
the putative genomic regions where the other known
resistance genes have been mapped, supporting the
hypothesis that MDR genes exist in wheat. For example,
DArT marker wPt-5769 on 3B identifed here signifcantly
associated with resistance to SB was also associated
with resistance to Puccinia triticina (Crossa et al., 2007).
Diversity Arrays Technology marker wPt-1159 on 3B,
which co-segregated with other DArT marker (wPt-4209)
Figure 4. Continued.
ADHI KARI ET AL.: QUANTI TATI VE RESI STANCE I N SPRI NG WHEAT 13
on 3B, was signifcantly associated with resistance to SB in
this study. Tis marker also was associated with powdery
mildew (Pm) and yellow rust (Yr) resistance and grain
yield (Crossa et al., 2007). Diversity Arrays Technology
marker wPt-8292 located at 110.8 cM on chromosome 4B
was signifcantly associated with resistance to BLS. Tis
same marker also was associated with QTL responsible for
grain yield (Crossa et al., 2007). Using wheat landraces and
DArT marker technology and AM approach in previous
studies (Adhikari et al., 2011b; Gurung et al., 2011) and
in this study, we have identifed the same genetic regions
or QTL associated with resistance to multiple pathogens.
Further work is needed to test if the same candidate gene
or diferent genes are involved in this MDR.
Te magnitude of LD and genomewide LD in
wheat can be afected by several factors such inbreeding
(McNally et al., 2009), selection (Palaisa et al., 2004),
domestication (Caicedo et al., 2007), outcrossing (McNally
et al., 2009), and admixture (Lexer et al., 2007). Diversity
is also reduced in hexaploid wheat as a consequence of the
polyploidy bottleneck (Akhunov et al., 2010). Although
gene diversity levels are similar in the A and B genomes, it
is greatly reduced in the D genome (Akhunov et al., 2010).
Te D genome also shows higher levels of LD than the A
and B genomes (Akhunov et al., 2010). Low recombination
is one of the factors afecting greatly uneven distribution
of diversity in the hexaploid wheat D genome compared
to the A and B genomes (Akhunov et al., 2010; Zhang et
al., 2010). In this study, varied levels of LD were observed.
As expected, the D genome had no markers or fewer
markers compared to the A and B genomes, suggesting
that map resolution can be improved in many regions of
chromosomes using the AM approach particularly for
chromosomes 3D, 4D, and 5D. Because of the low density
DArT markers on the array from the D genome (Diversity
Arrays Technology, 2011), it is likely that minor QTL
and interactions on the D genome chromosomes remain
undetected. Inadequate genome coverage may result in
failure to identify critical associations between markers
and traits (Flint-Garcia et al., 2003). Genetic distance
Figure 4. Continued.
14 THE PL ANT GENOME MARCH 2012 VOL. 5, NO. 1
used for LD estimations in this study was based on the
consensus map from several hexaploid wheat mapping
populations. A more precise estimation and improved
map resolution could be generated using highly abundant
SNP markers across wheat chromosomes or genomes. To
perform AM in a polyploid wheat genome, a larger panel
of genomewide SNP markers may be useful (Akhunov et
al., 2010; Chao et al., 2010).
In conclusion, the DArT marker system and AM
approach were useful for QTL discovery in spring wheat
landraces of diverse origin. Five and four genomic
regions were found to be associated with resistance to
BLS and SB, respectively. To utilize these genetic regions
in improved germplasm, the QTL should be validated
before deployment in breeding programs. High-
throughput genotyping technology such as genotype-by-
sequencing or SNP markers would be useful to develop
fner genetics maps and to identify more functional
diagnostic markers that are tightly linked to disease
resistances across the wheat accessions. Alternatively,
signifcant multiple loci can be selected and validated
by developing near-isogenic lines in diferent genetic
backgrounds and for testing them in multiple locations
or a targeted environment. Taken together, this emerging
knowledge would help identify genes involved in MDR
and enhance our understanding of molecular basis
of disease resistance in wheat and ultimately lead the
selection of resistance more likely to prove durable.
Supplemental Information Available
Supplemental material is available at http://www.crops.
org/publications/tpg.
Supplemental Table S1: Resistant wheat accessions to
bacterial leaf streak.
Supplemental Table S2: Resistant wheat accessions to
spot blotch.
Supplemental Table S3: Associations of wheat
accessions in diferent clusters.
Acknowledgments
Tis research was partly supported by the North Dakota Wheat
Commission, State Board of Agricultural Research and Education, North
Dakota, and USDA-ARS specifc cooperative agreement 58-5366-0-133.
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