DNA markers for Fusarium head blight resistance QTLs in two wheat populations

Abstract

Genetic resistance to Fusarium head blight (FHB), caused by Fusarium graminearum, is necessary to reduce the wheat grain yield and quality losses caused by this disease. Development of resistant cultivars
has been slowed by poorly adapted and incomplete resistance sources and confounding environmental effects that make screening
of germplasm difficult. DNA markers for FHB resistance QTLs have been identified and may be used to speed the introgression
of resistance genes into adapted germplasm. This study was conducted to identify and map additional DNA markers linked to
genes controlling FHB resistance in two spring wheat recombinant inbred populations, both segregating for genes from the widely
used resistance source ’Sumai 3’. The first population was from the cross of Sumai 3/Stoa in which we previously identified
five resistance QTLs. The second population was from the cross of ND2603 (Sumai 3/Wheaton) (resistant)/ Butte 86 (moderately
susceptible). Both populations were evaluated for reaction to inoculation with F. graminearum in two greenhouse experiments. A combination of 521 RFLP, AFLP, and SSR markers were mapped in the Sumai 3/Stoa population
and all DNA markers associated with resistance were screened on the ND2603/Butte 86 population. Two new QTL on chromosomes
3AL and 6AS wer found in the ND2603/Butte 86 population, and AFLP and SSR markers were identified that explained a greater
portion of the phenotypic variation compared to the previous RFLP markers. Both of the Sumai 3-derived QTL regions (on chromosomes
3BS, and 6BS) from the Sumai 3/Stoa population were associated with FHB resistance in the ND2603/Butte 86 population. Markers
in the 3BS QTL region (Qfhs.ndsu-3BS) alone explain 41.6 and 24.8% of the resistance to FHB in the Sumai 3/Stoa and ND2603/Butte 86 populations, respectively.
This region contains a major QTL for resistance to FHB and should be useful in marker-assisted selection.

Full text

Page 1

Abstract Genetic resistance to Fusarium head blight
(FHB), caused by Fusarium graminearum, is necessary
to reduce the wheat grain yield and quality losses caused
by this disease. Development of resistant cultivars has
been slowed by poorly adapted and incomplete resis-
tance sources and confounding environmental effects
that make screening of germplasm difficult. DNA mark-
ers for FHB resistance QTLs have been identified and
may be used to speed the introgression of resistance
genes into adapted germplasm. This study was conduct-
ed to identify and map additional DNA markers linked to
genes controlling FHB resistance in two spring wheat re-
combinant inbred populations, both segregating for
genes from the widely used resistance source ‘Sumai 3’.
The first population was from the cross of Sumai 3/Stoa
in which we previously identified five resistance QTLs.
The second population was from the cross of ND2603
(Sumai 3/Wheaton) (resistant)/ Butte 86 (moderately
susceptible). Both populations were evaluated for reac-
tion to inoculation with F. graminearum in two green-
house experiments. A combination of 521 RFLP, AFLP,
and SSR markers were mapped in the Sumai 3/Stoa pop-
ulation and all DNA markers associated with resistance
were screened on the ND2603/Butte 86 population. Two
new QTL on chromosomes 3AL and 6AS wer found in
the ND2603/Butte 86 population, and AFLP and SSR
markers were identified that explained a greater portion
of the phenotypic variation compared to the previous
RFLP markers. Both of the Sumai 3-derived QTL re-
gions (on chromosomes 3BS, and 6BS) from the Sumai
3/Stoa population were associated with FHB resistance
in the ND2603/Butte 86 population. Markers in the 3BS
QTL region (Qfhs.ndsu-3BS) alone explain 41.6 and
24.8% of the resistance to FHB in the Sumai 3/Stoa and
ND2603/Butte 86 populations, respectively. This region
contains a major QTL for resistance to FHB and should
be useful in marker-assisted selection.
Keywords Wheat · Scab · QTL mapping ·
Disease resistance · Fusarium graminearum
Introduction
Fusarium head blight, caused by Fusarium graminearum
Schwabe [telomorph: Gibberella zeae Schw.(Petch)], is
recognized as one of the most-destructive diseases of
wheat. Due to quantitative inheritance and difficulties in
screening for the presence of resistance genes (Bai and
Shaner 1994), DNA markers associated with such genes
may increase the efficiency of selecting for resistance.
Previously, from analysis of a recombinant inbred popu-
lation from the cross ‘Sumai 3’/‘Stoa’, we identified DNA
markers for a major FHB resistance QTL on chromosome
3BS (Waldron et al. 1999). This QTL, designated
Communicated by B.S. Gill
J.A. Anderson (✉) · S. Liu
Department of Agronomy and Plant Genetics, 411 Borlaug Hall,
University of Minnesota, St. Paul, MN 55108, USA
e-mail: ander319@tc.umn.edu,
Tel. +1-612-625-9763, Fax: +1-612-625-1268
R.W. Stack
Plant Pathology Department, Walster Hall,
North Dakota State University, Fargo, ND 58105-5051, USA
J. Mitchell Fetch
Cereals Research Centre, Agriculture and Agrifood Canada,
Winnipeg, MB R3T-2M9, Canada
B.L. Waldron
USDA-ARS Forage and Range Research Laboratory,
Logan, UT 84322-6300, USA
A.D. Fjeld · C. Coyne
USDA-ARS, Pullman, WA 99164-6420, USA
B. Moreno-Sevilla
Western Plant Breeders, 6025 West 300 South, Lafayette,
IN 47905, USA
Q.J. Song · P.B. Cregan
USDA-ARS, Building 006, Room 100, Beltsville, MD 20705,
USA
R.C. Frohberg
Plant Science Department, Loftsgard Hall,
North Dakota State University, Fargo, ND 58105-5051, USA
Theor Appl Genet (2001) 102:1164–1168© Springer-Verlag 2001
ORIGINAL ARTICLE
J.A. Anderson · R.W. Stack · S. Liu · B.L. Waldron
A.D. Fjeld · C. Coyne · B. Moreno-Sevilla
J. Mitchell Fetch · Q.J. Song · P.B. Cregan
R.C. Frohberg
DNA markers for Fusarium head blight resistance QTLs
in two wheat populations
Received: 17 August 2000 / Accepted: 16 October 2000

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Qfhs.ndsu-3BS, was derived from the Chinese cultivar
Sumai 3, a widely used resistance source for this disease.
Four other QTLs associated with resistance were identi-
fied, two of which were derived from Sumai 3. Bai et al.
(1999) identified a major QTL for FHB resistance in
‘Ning7840’, which is believed to have inherited its scab re-
sistance from Sumai 3 (Liu and Wang 1990). Contrary to
the original tentative location of this QTL on chromosome
7B (Bai et al. 1999), this gene is believed to reside in the
Qfhs.ndsu-3BS region (Bai, personal communication).
Exploitation of marker-QTL linkages in breeding pro-
grams requires robust markers that are effective across
genetic backgrounds. A further requirement is that such
diagnostic approaches provide efficiencies compared
with conventional, phenotype-based procedures. There
are few documented cases of using DNA markers for
QTLs in applied plant breeding programs (Young 1999).
A contributing factor to this failure is that the QTLs often
show G×E effects, or their effects in new genetic back-
grounds are less pronounced (Tanksley and Nelson 1996).
The objectives of the present research were to verify
the FHB QTLs identified in the Sumai 3/Stoa population
with another population and obtain more closely linked
markers to Qfhs.ndsu-3BS.
Materials and methods
FHB screening
A population of 112 F5-derived recombinant inbred lines (RIL) from
the cross Sumai 3 (resistant)/Stoa (mod. susceptible) grown in the
greenhouse was evaluated for reaction to inoculation with conidia
from F. graminearum in two experiments, as described by Waldron
et al. (1999). A population of 139 F5-derived recombinant inbred
lines (RIL) from the cross ND2603 (Sumai 3/Wheaton) (resis-
tant)/Butte 86 (moderately susceptible) was evaluated for reaction to
inoculation with conidia from F. graminearum in two greenhouse ex-
periments, as described by Mitchell Fetch et al. (1998). Briefly, at
anthesis, an average of nine spikes of the same size and maturity in
each of three replications per RIL were inoculated with a 10-µl drop-
let (50,000 conidia/ml) of conidial suspension placed directly into a
single spikelet near the center of the spike, following procedures de-
scribed by Stack (1989). The conidial suspension contained a mix-
ture of three F. graminearum isolates in all experiments except for
the first Sumai 3/Stoa which used experiment a single isolate. This
procedure bypasses primary infection and targets Type II resistance
(Schroeder and Christensen 1963; Wang and Miller 1988). A gentle
overhead mist was applied and plants were covered with a plastic
humidity tent for three nights following inoculation. Three weeks
after inoculation, spikes were scored individually for visual symp-
toms on a 0–100% FHB severity scale (Stack and McMullen 1985).
For both populations, the entry means of the two experiments that
included all RI lines were used in subsequent analyses.
DNA marker analysis
RFLP mapping in the Sumai 3/Stoa population was described by
Waldron et al. (1999). Only those RFLP markers significantly as-
sociated (P<0.05) with FHB on the Sumai 3/Stoa population were
screened for polymorphism and mapped in the ND2603/Butte 86
population.
Because of difficulties in obtaining linked markers for genomic
regions significantly associated with FHB resistance (e.g., 3BS),
AFLP analysis was initiated to target markers to these regions using
a selective genotyping approach. We selected ten RI lines (five resis-
tant and five susceptible) based on the FHB reaction of lines and/or
the RFLP genotype of markers in the target region. We used the
AFLP kits from Gibco, BRL with slight modifications from the man-
ufacturer’s instructions. Primer pairs revealing segregation that sug-
gested association with resistance or linkage to the intended RFLP
marker in the ten RILs composing the selective genotyping array
were used to genotype all members of the population. All other poly-
morphic fragments revealed by such primer pairs were mapped on
the entire population. Only one pair of AFLP primers (E40M59) was
mapped in the ND2603/Butte 86 population. The AFLP markers are
designated according to the nomenclature of McIntosh et al. (1998).
Primers for all microsatellites (SSRs) published by Röder et al.
(1998) were synthesized and screened for polymorphism among
the four parents of these populations. Markers known to be located
in putative QTL regions based on our previous research were giv-
en the highest priority. PCR amplification was as described by
Röder et al. (1998) except that 35 cycles of amplification were
used instead of 45. Visualization of fragments was by electropho-
resis in 5% polyacrylamide gels and silver staining according to
the protocol of Bassam et al. (1991). Polymorphic markers were
mapped in both populations. Fifty microsatellites, designated with
the prefix ‘BARC’, were kindly provided by Q.J. Song and
P. Cregan, USDA-ARS, Beltsville, Md. These were screened for
polymorphism in the parents and aneuploid stocks to identify
those located on chromosome 3BS. All such polymorphic markers
on chromosome 3BS were used to screen the populations.
Linkage maps were constructed using MAPMAKER Macintosh
v2.0 (Lander et al. 1987) and aneuploid analysis was used to deter-
mine the chromosomal arm location of markers in linkage groups as
described by Anderson et al. (1992). Markers were subjected to re-
gression and interval analysis using the computer program QGENE
(Nelson 1997) to identify significant (P<0.01) associations between
individual DNA markers and FHB resistance. Multiple regression
models were developed by initially including the single best marker
in all QTL regions and eliminating those that were not significant at
P <0.01 in the model one at a time. Two-way epistatic interactions
involving one of the significant markers were calculated using all
markers mapped in the respective population.
Results and discussion
The FHB data for the Sumai 3/Stoa cross was described in
Waldron et al. (1999). The ND2603/Butte 86 population
displayed a normal distribution, transgressive segregants,
and significant variation among RILs for FHB severity
(Table 1, Fig. 1). Heritability on an entry mean and plot
basis was 0.50 and 0.24, respectively. A large component
(34%) of the phenotypic variance for FHB severity in the
ND2603/Butte 86 population was the RI line×experiment
interaction (Table 1). This source of variation accounted
for only 17% of the variation in the Sumai 3/Stoa popula-
tion (Waldron et al. 1999). This result emphasizes the im-
portance of multi-experiment data for this trait.
In addition to the 360 RFLP loci mapped by Waldron et
al. (1999) in the Sumai 3/Stoa population, we mapped 151
loci resulting from 16 pairs of AFLP primers and ten
SSRs. The AFLP primer sets were chosen because each
had at least one polymorphic fragment that was suggestive
of being associated with FHB resistance based on the se-
lective genotyping array of ten individuals. However, such
fragments, when mapped on the entire population, were no
more likely to be associated with QTLs than other frag-
ments that segregated independent of FHB reaction in the
ten individuals (data not shown). Four genomic regions
containing putative quantitative trait loci (QTLs) were as-

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sociated (P<0.01) with FHB resistance from the combined
analysis of two experiments, two from Sumai 3 and two
from Stoa (Table 2). The two major QTLs on chromo-
somes 3BS and 2AL, and a minor QTL on chromosome
4BS, were identified by Waldron et al. (1999). The marker
Xgwm493 is a SSR significantly associated with FHB re-
sistance with an R2value of 41.6% (Table 2). This locus
mapped in the Qfhs.ndsu-3BS region identified by Waldron
et al. (1999) and is now the most-strongly associated with
FHB resistance in this population. The mapping of AFLPs
on chromosome 6BS linked the two minor QTLs reported
by Waldron et al. (1999) approximately 24-cM apart. Be-
cause neither QTL has a strong or consistent effect in the
two environments, we are considering this a single, minor
QTL region. A multiple regression model was developed
that included all of the markers significantly associated
with FHB resistance and listed in Table 2 with the excep-
tion of XBARC101 (Table 3). This model explained 54% of
the variation for FHB resistance in this population. Given
the heritability on an entry mean basis of 0.78 for FHB re-
sistance in this population (Waldron et al. 1999), we be-
lieve that these markers represent all major QTLs segregat-
ing in this population. It is likely that other minor QTLs
and/or epistatic interactions have not yet been identified.
A total of 62 loci representing all Sumai 3/Stoa QTL re-
gions were mapped in the ND2603/Butte 86 population.
Encouragingly, both QTLs associated with FHB resistance
from Sumai 3 in the Sumai 3/Stoa population were also as-
sociated with resistance in the ND2603/Butte 86 population
(Table 2). This is an important step in verifying the effec-
tiveness of these markers in other genetic backgrounds.
Two QTL from ND2603, on chromosomes 3AL, and 6AS
were also discovered in this population. Neither of the two
Stoa-derived QTLs were associated with FHB resistance in
the ND2603/Butte 86 population (Table 2). The multiple
regression model explained 34% of the variation for FHB
resistance in this population and included all of the signifi-
cant markers listed in Table 2 except Xbcd1383 (Table 3).
No epistatic interactions were found in either population
that increased the percent of variation explained by more
than 5%. Therefore, these interactions are minor in effect
Table 1 ANOVA table and
proportion of phenotypic varia-
tion for Fusarium head blight
from recombinant inbred lines
from the cross ND2603/
Butte 86
Sourcea
df
MS
F-ratio
P
Proportion of
phenotypic
varianceb
Experiments1 35,62165.22 0.013
Replications
(in experiments)
4546 2.820.025
RI lines
RI lines×experiments
Error
138 1,3131.98 <0.001 0.24
138662 3.41<0.0010.34
546 1940.42
Total 827
aExperiments and Replications
were considered to have ran-
dom effects, and RI lines fixed
effects
bHeritability was 0.50 and 0.24
on an entry mean and per plot
basis, respectively
Table 2 Coefficients of determination and P values for DNA markers associated with Fusarium head blight resistance in the Sumai
3/Stoa and ND2603/Butte 86 recombinant inbred populations
Markera
ChromosomeSource of
resistance allelea
Sumai 3/StoaND2603/Butte 86
R2×100
PR2×100
P
Xgwm493/Xgwm533
XksuH16
XksuH4
XBARC101/Xbcd1383
Xwg909
Xbcd941
3BS
2AL
6AS
6BS
4BSb
3AL
Sumai 3/ND2603
Stoa
Sumai 3/ND2603
Sumai 3/ND2603
Stoa
ND2603
41.6
14.3
1.0
9.2
7.2
0.1
<0.001
<0.001
0.32
0.001
0.007
0.48
24.8
0.0
11.6
4.9
0.1
9.1
<0.001
0.66
<0.001
0.009
0.28
<0.001
aMarkers and resistance sources to the left of the slash refer to the
Sumai 3/Stoa population and to the right refer to the ND2603/
Butte 86 population
bThis marker was reported as being located on 4BL by Waldron
et al. (1999). Due to the position of new markers mapped in this
region, the arm position of this marker is believed to be 4BS
Fig. 1 Histogram of mean percentage of Fusarium head blight
severity for parents and 139 RIL lines from the cross ND2603/
Butte 86

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Table 3 Multiple regression models of DNA markers to explain
variation in reaction to Fusarium head blight in two spring wheat
populations
SourceEstimatea
Standard error
P
Sumai 3/Stoab
Xgwm493
Xwg909
XksuH16
ND2603/Butte 86c
Xgwm533
Xbcd941
XksuH4
–10.32
5.35
4.73
1.24
1.17
1.24
<0.001
<0.001
<0.001
–5.98
–3.62
–3.60
1.11
1.08
1.09
<0.001
0.001
0.001
aRegression coefficients in the multiple regression models. Posi-
tive or negative numbers indicate the direction of the response.
Negative numbers indicate lower FHB severity derived from the
female parent, either Sumai 3 or ND2603
bThe coefficient of determination (R2) was 0.54
cThe coefficient of determination (R2) was 0.34
Fig. 2 Interval analysis of data
for chromosome 3B for Fusari-
um head blight resistance in the
Sumai 3/Stoa and ND2603/
Butte 86 recombinant inbred
populations. The dark contour
in each map represents the
mean of the two experiments.
The two lighter colored con-
tours represent individual ex-
periments. The two maps are
aligned at the Xgwm493 locus.
Xgwm389 and Xgwm533 were
also mapped in both popula-
tions
compared to the individual effects of the QTLs identified,
and, therefore, were not included in the multiple regression
models.
Chromosomes 3B and 6B have been implicated in
FHB resistance by monosomic and/or backcross recipro-
cal monosomic analysis in other wheat germplasms, but
not in studies published to-date using Sumai 3 [see
Buerstmayr et al. (1999) for a review]. We consider the
discovery of QTLs for FHB resistance on chromosomes
3B and 6B using DNA markers in two segregating popu-
lations to constitute strong evidence that these represent
important genomic regions that in large part are respon-
sible for the resistance observed in Sumai 3. The most
significant genomic region associated with FHB resis-
tance in our populations is located on the short arm of
chromosome 3B. Interval analysis revealed a peak LOD
score of 13.8 and 8.2 for this region in the Sumai 3/Stoa
and ND2603/Butte 86 populations, respectively (Fig. 2).
The RFLP map of Waldron et al. (1999) gave a peak
LOD score of 4.4 for this region. The observation that
the multiple QTLs seem to have additive effects, as indi-
cated by the fact that all but one in each population re-
tain significance in multiple regression models, means
Fig. 3 DNA fragments from amplification of wheat genotypes
with the microsatellite marker gwm533. Lanes 1, 14, and 27 are
10-kb ladder marker lanes; 2, 3, 15, and 16 are parents of the pop-
ulations; 4–13 and 17–26 are random subsets of progeny from the
Sumai 3/Stoa and ND2603/Butte 86 populations, respectively. The
fragments centered at 145 and 119 bp are alleles of the
Xgwm533.1 and Xgwm533 loci in the Sumai 3/Stoa and ND2603/
Butte 86 populations, respectively. The fragments between 95 and
100 bp represent the Xgwm533.2 locus in the Sumai 3/Stoa popu-
lation. This locus did not segregate in the ND2603/Butte 86 popu-
lation

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that presumably they can be combined to give greater
levels of FHB resistance. However, the effect of
Qfhs.ndsu-3BS is so large in comparison to the other
QTLs (almost twice the effect of the next best QTL in
the multiple regression model) that this appears to be the
major resistance gene segregating in these populations.
After mapping all of the published SSR markers from
chromosome 3BS (Röder et al. 1998) that are polymor-
phic in both populations, we have positioned Qfhs.ndsu-
3BS between Xgwm493 and Xgwm533 (Fig. 3). Selection
for the region bracketed by these two markers greatly
skews both populations toward more-resistant types
(Fig. 4). This bodes well for the exploitation of these
markers as bracketing markers for the QTL which can be
used for marker-assisted selection, thereby accelerating
the development of resistant cultivars. Future research
will include mapping additional markers on 3BS, verify-
ing these and other new markers in additional popula-
tions, and initiating marker-assisted selection using
markers on 3BS in our wheat breeding program.
Acknowledgments We thank Justin B. Hegstad and Emily J.
Wennerlind for technical assistance, and J.C. Nelson for providing
the QGENE software. Research supported by USDA-NRI grant
96–35300–3721 to J.A.A., the Millers’ National Federation, North
Dakota State University Research Foundation, North Dakota Agri-
cultural Experiment Station, USDA-ARS, the Minnesota Agricul-
tural Experiment Station, and the U.S. National Scab Initiative
(via USDA-ARS). These experiments comply with the current
laws of the United States of America.
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Fig. 4 Histograms of Fusarium head blight severity for RI lines
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