Euphytica 139: 167–172, 2004.
C ?2004 Kluwer Academic Publishers. Printed in the Netherlands.
DNA and morphological markers for a Russian wheat aphid resistance gene
A. Arzani1,2,∗, J.H. Peng1& N.L.V. Lapitan1
1Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO 80523, U.S.A.;2Agronomy
and Plant Breeding Department, College of Agriculture, Isfahan University of Technology, Isfahan 84156, Iran
(∗author for correspondence: e-mail: a email@example.com)
(Received 4 February 2004; accepted 26 August 2004)
Key words: glume colour, marker-assisted selection, microsatellites, resistance, Russian wheat aphid, wheat
The Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), is a significant insect pest of wheat worldwide.
Morphological and molecular markers associated with RWA resistance could be used to increase the accuracy and
efficiency of selection of resistant germplasm and facilitate transfer to desirable wheat genotypes. The objective of
this work was to identify microsatellite (SSR) markers linked to the RWA resistance gene (Dn4) and glume-colour
hexaploid-11) and a resistance cultivar (Halt). Two microsatellite markers Xgwm106 and Xgwm337 flanked Dn4 on
the short arm of chromosome 1D at 5.9 and 9.2 cM, respectively. Two other microsatellite markers, Xpsp2999 and
Xpsp3000, at the distal part of this chromosome arm are linked to Dn4 and to Rg2. The accuracy and efficiency of
marker-assisted selection were calculated for homozygous Dn4Dn4 genotypes in the F2generation. The gene Rg2
for red glume colour can also be used for marker-assisted selection of Dn4 gene individually and in combination
with microsatellite markers. When used together, the closest markers Xgwm106 and Xgwm337, provide 100%
accuracy and 75% efficiency. One hundred percent accuracy is also achieved when the morphological marker red
glume is used in combination with either Xgwm106 or Xgwm337. Using these flanking markers, it may be possible
to fix resistance to RWA in the first segregating generation of an F2population without infestation with aphids.
The Russian wheat aphid (RWA), Diuraphis noxia
(Mordvilko) (Homoptera: Aphididae), is one of the
most harmful pests of wheat in the world (Kovalev
has become a major economic pest of wheat (Triticum
aestivum L.) and barley (Hordeum vulgare L.) in the
western United States (Legg & Amosson, 1993). Con-
trol of the RWA with insecticides is neither environ-
mentally nor economically effective because infested
susceptible plants display leaf rolling, which shelters
the aphids from the insecticides (Baker & English,
1988; Ma et al., 1998). Deployment of resistant cul-
tivars is the most economical and environmentally safe
method to eliminate the use of insecticides and to re-
duce crop losses due to this pest.
DNA markers have been developed for nine genes
conferring resistance to RWA. These genes include
Dn1, Dn2, Dn4, Dn5, Dn6, Dn7, Dn8, Dn9, and Dnx
(Anderson et al., 2003; Botha et al., 1995; Liu et al.,
2001, 2002; Miller et al., 2001; Myburg et al., 1998).
The use of DNA markers in breeding for RWA resis-
tance provides several advantages including the abil-
ity to screen year-round instead of only during cooler
months of the year when the RWA multiplies, and abil-
ity to breed for resistance in places where the aphid
does not yet occur. The most important application
of marker-assisted-selection (MAS) for RWA is the
potential to pyramid two or more resistance genes in
the same cultivar. In the United States, only one bio-
type existed following its first detection in 1986 until
the spring of 2003, when a new biotype appeared in
Colorado (Haley et al., 2004). The new biotype (bio-
type B) was virulent to Dn4 and eight other known
resistance genes in wheat. However, the old biotype
(biotype A) is still widespread, and was observed to
occur on the same plants as the new biotype (Peairs,
personal communication). This creates a greater need
to combine resistance genes for the new biotype with
resistance genes for the old biotype. When combining
pending on the genetic distance between the markers
and the resistance genes.
The first RWA resistant cultivar released in the US
gene was subsequently used extensively for breeding
in the Colorado State University (CSU) wheat breed-
ing program and was introduced into five other genetic
backgrounds (Haley et al., 2003; Quick et al., 2001).
A molecular map for Dn4 was published by Ma et al.
(1998). The map contained two flanking RFLP mark-
ers, Xabc156-1D and Xksud14-1D, with genetic dis-
tances of 32.5 and 11.6 cM, from Dn4, respectively.
These markers were not deployed in the wheat breed-
ing program, primarily because of the large genetic
distances from the gene. Tests showed that these spe-
cific markers were not diagnostic for Dn4, in that they
did not always distinguish susceptible cultivars from
resistant cultivars containing Dn4 (Erker, 1998). Fur-
thermore, RFLPs are not efficient for MAS, because
it is expensive, labour-intensive and require radioiso-
topes for detection.
1990s and were shown to have a high polymorphism
index (Bryan et al., 1997; R¨ oder et al., 1998). Mi-
tively detect polymorphisms between wheat cultivars
and are PCR-based (Plaschke et al., 1995; R¨ oder et al.,
lite markers for Dn4. Two microsatellite markers iden-
tified in this study, Xgwm106-1D and Xgwm337-1D,
were also reported to be linked to Dn4 by Liu et al.
(2002). Two other microsatellite markers are reported
here along with a morphological marker (red glume).
ers for MAS are presented.
Materials and methods
One hundred ten F2-derived F3 (F2:3) families
originating from a cross between a susceptible
MYT, Mexico (T. turgidum-D67.2/P66.270//Aegilops
tauschii) and the resistant cultivar ‘Halt’ were used in
this study. SH-11 was selfed five times followed by
testing for RWA reaction before being used as a par-
ent in this cross. The self-pollinated progenies of each
individual F2(F2:3and F2:4families) were planted in
the CSU insectary and used for RWA phenotypic as-
sessments. F2-derived F3families were used for DNA
RWA resistance evaluation
Seedling reaction to the RWA biotype A was studied
during two seasons (November–December 2000 and
2001) using F2:3families. F2-derived F4families were
evaluated for RWA reaction during February–March
2002. Parents, 110 F3(2000 and 2001) or F4(2002)
families and check cultivars (‘Carson’, ‘TAM 107’
(susceptible) and ‘Halt’ (resistant)) were planted in a
3:2:1:1 (v/v/v/v) soil mix consisting of soil, peat moss,
vermiculite, and perlite in 25 cm × 50 cm flat trays
(12 rows). Each row contained 15 plants with 4-cm
spacing between rows. Row number six of each tray
was divided to three parts and allocated to three
check cultivars. A randomized complete block design
with four replications (two replicates per year) was
used. The trays were uniformly watered and fertilized
throughout the experiment. The seedlings were
grown in the greenhouse under a 16 h photoperiod at
25 ± 2◦C.
screening. Seedlings were infested with five RWA per
plant at the one-leaf stage. RWA damage (leaf rolling
and leaf chlorosis) was scored according to Nkongolo
et al. (1991). Seedling damage was measured on a 1–9
scale, 1 denoting healthy plants and 9 denoting dying
or dead plants. Symptom expression of susceptible
and resistant seedlings was recorded when the suscep-
tible check showed severe leaf rolling and chlorotic
streaking (scores of 8–9 after 21–28 days). Seedlings
with chlorotic spots caused by aphid feeding and
without leaf rolling (scores 1–4), and seedlings with
chlorotic streaking and leaf rolling (scores 5–9) were
recorded as resistant and susceptible, respectively.
Based on the reaction of F3 families to RWA, the
genotypes of the corresponding F2 individuals were
determined: homozygous resistant (RR, all plants in
the F3family were resistant); heterozygous resistant
(Rr, most of the plants in the F3family were resistant
and the minority were susceptible); and homozygous
susceptible (rr, all plants in the F3 family were
Chi-square tests for goodness of fit to phenotypic
segregation ratios of 3:1 (resistant:susceptible) and
genotypic segregation ratios of 1:2:1 (homozygous
resistant:heterozygous:homozygous susceptible) were
used to determine the mode of inheritance of the RWA
Glume colour phenotyping
The same population of F2-derived F3(F2:3) families
colour was scored at maturity as either red or white for
the plants within families. Families were then classi-
fied into homozygous red, heterozygous and homozy-
gous white classes. Chi-square tests for goodness of fit
to phenotypic segregation ratios of 3:1 (red:white) and
mine the mode of inheritance of the glume colour trait.
Red colour was associated with RWA susceptibility.
DNA isolation and microsatellite analysis
Genomic DNA was extracted from young leaf tissue
according to Ma & Sorrells (1995). DNA sequences
(Bryan et al., 1997; R¨ oder et al., 1998) were ob-
tained from M. R¨ oder (IPK, Gatersleben, Germany),
M. Gale (John Innes Institute, Norwich, UK), and
Primers were synthesized by Gene Link, Inc., USA
ers (Xgwm33, Xgwm106, Xgwm337 and Xgwm458,
Xpsp2999, Xpsp3000, Xbarc149 and Xbarc150) were
screened for polymorphisms between the parents, and
between bulked DNA of six homozygous-resistant F3
families and six susceptible F3families. Microsatel-
lites showing polymorphisms were used to genotype
The PCR reaction was performed in a 25-µl vol-
ume using a PTC-200 MJ thermocycler (MJ Research,
Inc., Waltham, MA). The reaction mixture contained:
1 U of Taq-polymerase and 100 ng of template DNA.
After 3 min of denaturation at 94◦C, 45 cycles were
(based on primer annealing temperature), 2 min at
amplified products of Xgwm33, Xgwm106, Xgwm458
sis in 3% agarose gels (Fisher Biotech) at 4 V/cm in
1 × TBE buffer. PCR amplified products of Xgwm337,
8 M urea) at 150 V for 2.5 h in 1 × TBE buffer. Gels
banding patterns were visualized using Alpha Imager
(Alpha Innotech Corporation, San Leandro, CA).
Linkage analysis and evaluation of markers
Mapping analysis was conducted using MAP-
MAKER/EXP version 3.0b (Lander et al., 1987;
Lincoln et al., 1992). The Kosambi mapping function
fractions into map distances (cM).
The effectiveness of markers for MAS was evalu-
ated by calculating accuracy and efficiency using the
empirical formula proposed by Peng et al. (2000). Ac-
curacy of a marker is calculated by the number of ho-
mozygous resistant plants among the total number of
that were homozygous for the marker among the ho-
mozygous resistant plants multiplied by 100.
Results and discussion
Inheritance of RWA resistance and red glume colour
Among the tested F2:3families from the cross ‘Halt’
(R) × synthetic hexaploid-11(S), 14 families were
homozygous resistant, 66 heterozygous, and 27 ho-
mozygous susceptible. Chi-square analysis fit a phe-
notypic segregation ratio of three resistant:one suscep-
tible (χ2= 0.0031, p = 0.95). This result is in agree-
ment with previous studies reporting the presence of
a single dominant gene Dn4 in ‘Halt’ (Quick et al.,
1996). Dn4 was derived from PI 372129 and was also
shown by Ma et al. (1998) to segregate in a domi-
nant fashion. Note however that the data did not fit the
expected-genotypic segregation ratio of 1RR:2Rr:1rr
gotes than expected. Significant deviation from ex-
pected Mendelian segregation ratios of 3:1 for Dn4
locus has been reported by Liu et al. (2002) for one of
their populations (PI 372129, (Dn4)/‘Thunderbird’).
‘Halt’ had white glume colour. The segregation of this
56 heterozygous, and 27 homozygous white glume.
2df= 9.3, p ≤ 0.01). There were more heterozy-
Figure 1. DNA bands amplified from leaves of F2-derived F3families of synthetic hexaploid-11 (susceptible parent, Sp) × ‘Halt’ (RWA
resistant parent containing Dn4, Rp), using microsatellite Xgwm337 marker and electrophoresed in a 7% polyacrylamide gel. L = 50 bp ladder,
R: homozygous resistant, S: susceptible, H: heterozygous F3families (F2plants); about 110 F2:3families were evaluated.
Chi-square analysis fitted a genotypic segregation ra-
tioof1:2:1(χ2= 0.547, p = 0.75).Thisresultagreed
with previous studies showing the presence of a single
dominant gene for red glume colour (Rg2) in chromo-
some 1DS (Jones et al., 1990).
Linkage map of Dn4 and Rg2
Of the eight microsatellite markers tested, four
(Xgwm106, Xgwm337, Xpsp2999 and Xpsp3000) de-
tected polymorphisms between the resistant and sus-
ceptible parents and between bulked DNA of resistant
and susceptible individuals. A 125 bp fragment was
detected in ‘Halt’ by the marker Xgwm106, but was
absent in the susceptible parent synthetic hexaploid-
11’ line. The marker Xgwm337 showed a polymor-
phic pattern displaying a 225-bp band amplified from
the DNA of ‘Halt’, and a 250-bp DNA fragment spe-
cific to synthetic hexaploid-11 (Figure 1). The marker
Xpsp2999 detected a polymorphic pattern displaying a
125-bp fragment characteristic of ‘Halt’, and a 150-bp
DNA fragment specific to synthetic hexaploid-11. The
105-bp band amplified from ‘Halt’, and a 130-bp DNA
fragment specific to synthetic hexaploid-11.
Each of the microsatellite markers was linked to
Dn4 and Rg2 (Figure 2). The closest markers linked
to Dn4 were Xgwm106 and Xgwm337, with genetic
distances of 5.9 and 9.2 cM, respectively. The same
markers were shown to be linked to Dn4 by Liu
et al. (2002) in populations with different parents (PI
present only in the resistant parent, was mapped for
Xgwm106. For Xgwm337, the bands reported by Liu
et al. (2002) (175 and 195 bp, for resistant and suscep-
tible parents, respectively) were slightly smaller than
those observed here. While the previous study used
a 2% gel in separating bands, we used a 7% poly-
acrylamide (Figure 1). In both studies, the larger band
corresponded to the susceptible parent. The mapped
markers on wheat chromosome 1DS.
bands are likely the same in the two studies. Liu et al.
(2002) reported genetic distances from Dn4 of 7.4 and
12.9 cM, for Xgwm106 and Xgwm337, respectively.
Two other microsatellite markers, Xpsp2999 and
Xpsp3000, were also linked to Dn4 at distances of 36.5
and 39 cM, respectively. In addition, Rg2 was linked to
Dn4 at 27 cM. Jones et al. (1990) reported that Gli-D1
on the short arm of chromosome 1D is tightly linked
to Rg2 and Lr21 (1.4 and 5.6% recombination, respec-
tively) in the following order: Gli-D1-Rg2-Lr21. The
Lr21 gene was recently cloned by a map-based ap-
proach and shown to be contained in RFLP marker
linkage between XksuD14 and Dn4 at a distance of
32.5 cM on the short arm of chromosome 1D. Rg2 was
mapped as a QTL on chromosome 1DS (B¨ orner et al.,
2002), and was found to be in a highly comparable po-
sition with the Bg and Rg1 genes on chromosomes 1A
and 1B, respectively.
Marker-assisted selection for RWA resistance
Since our interest was to find markers that can be used
effectively for marker assisted selection of the Dn4
gene, percent accuracy and efficiency for the identified
markers was calculated (Tables 1 and 2). For single
markers, Xgwm337 gave the highest values for accu-
racy and efficiency. Although Xgwm106 is closer to
Dn4, it is a dominant marker and precludes distinction
Table 1. Accuracy and efficiency of marker-assisted selection of ho-
mozygous resistant genotypes of the Dn4 gene based on a single
linked marker in the F2generation
MarkerAccuracy (%) Efficiency (%)
9.2 81.3 83.9
aDominant marker, homozygotes not distinguished from heterozy-
Table 2. Accuracy and efficiency of marker-assisted selection of
homozygous resistant genotypes of the Dn4 gene based on two
flanking/non-flanking markers in the F2generation
Map distance (cM)
between flanking (F)
Marker or marker
Xgwm337-Xgwm106 F15.1 100.0 75.8
Xgwm337-Xpsp2999 F45.6 91.048.1
Xgwm106-Xpsp2999 NF 30.591.052.4
Xgwm106-Xpsp3000 NF 33.086.349.8
Xpsp2999-Xpsp3000 NF2.554.6 49.8
F 36.3 100.056.5
NF 21.2 100.0 62.3
NF 11.8 42.949.8
between homozygotes and heterozygotes. The values
for Xgwm337 indicate that 81% of plants selected with
the 225 bp band will also be resistant, and that 84%
of plants homozygous for the 225 bp band will be ho-
mozygous resistant. Based on phenotypic test results,
the maximum accuracy and efficiency for selection of
homozygous resistant genotypes of Dn4 in the F2gen-
eration is 33.33% because only one-third of the re-
sistant genotypes are homozygous and the other two-
thirds are heterozygous (Anderson et al., 2003; Peng
et al., 2000).
It is generally accepted that the accuracy and effi-
ciency of MAS will be improved if, rather than a sin-
gle marker, two markers flanking the target gene are
used. Table 2 shows that the accuracy and efficiency of
identifying homozygous resistant genotypes of Dn4 is
improved when two flanking markers are used. When
used together, the closest markers to Dn4, Xgmw106
and Xgwm337, provide 100% accuracy and 76% effi-
ciency. One hundred percent accuracy is also achieved
either Xgwm106 or Xgwm337 (Table 2). The use of a
visible and easily scorable marker such as red glume
is an advantage. In our studies, we were able to dis-
tinguish homozygous Dn4 genotypes from heterozy-
gotes based on visual observation of glume color. Het-
erozygotes for Rg2 showed a mixture of red and white
glumed plants in the same family.
(2000) who reported an accuracy of 70% for predict-
when the map distance between two flanking markers
was 81.9 cM. Using the SSR flanking markers, it may
ing generation of an F2population without infestation
with aphids (Anderson et al., 2003).
We thank Isfahan University of Technology, Iran, for
A. Arzani’s fellowship support. We thank Frank Peairs
and Jeff Rudolph for providing Russian wheat aphids
and for use of their facilities at the CSU Insectary. This
34205-6375, USDA Contract No. 2001-52100-11293,
and Hatch Funds.
Anderson, G.A., D. Papa, J.H. Peng, M. Tahir & N.L.V. Lapitan,
2003. Genetic mapping of Dn7, a rye gene conferring resistance
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