RESEARCH
Identification of Novel QTL
for Sawfly Resistance in Wheat
J. D. Sherman, D. K. Weaver, M. L. Hofland, S. E. Sing, M. Buteler, S. P. Lanning,
Y. Naruoka, F. Crutcher, N. K. Blake, J. M. Martin, P. F. Lamb, G. R. Carlson, and L. E. Talbert*
ABSTRACT
The wheat stem sawfly (WSS) (Cephus cinctus
Nort.) is an important pest of wheat (Triticum aestivum L. em. Thell.) in the Northern Great Plains. This
paper reports the genetic analysis of antixenosis
for egg-laying WSS females in recombinant inbred
lines (RIL) of hard red spring wheat. Female WSS
preferentially choose certain wheat genotypes for
egg-laying, with the cultivar Reeder being preferred
and Conan being less preferred. We measured
percent stem infestation and percent stem cutting for 91 RIL from a Reeder–Conan cross in four
sawfly-infested locations in Montana. Heritability
based on means over environments was h2 = 0.86
for infestation and h2 = 0.75 for cutting. Percent
infestation was negatively correlated with heading date (r = −0.57, P < 0.001) and degree of stem
solidness (r = −0.31, P < 0.01). A molecular map
was created with 431 markers. Quantitative trait
loci (QTL) for infestation and cutting were identified
as cosegregating with QTL for heading date (controlled by Ppd-D1 on chromosome 2D) and stem
solidness (controlled by Qss.msub.3BL). Additionally, significant QTL for infestation and cutting on
chromosomes 2D and 4A were present in several
environments, and did not cosegregate with heading date, plant height, or solid stems. These QTL
may complement the use of solid stems for host
plant resistance by developing wheat lines that
vary for attractiveness to the wheat stem sawfly.
J.D. Sherman, S.P. Lanning, Y. Naruoka, N.K. Blake, J.M. Martin, and
L.E. Talbert, Plant Sciences and Plant Pathology Dep., Montana State
Univ., Bozeman, MT 59717; D.K. Weaver, M.L. Hofland, S.E. Sing,
and M. Buteler, Land Resources and Environmental Sciences Dep.,
Montana State Univ., Bozeman, MT 59717; F. Crutcher, Plant Pathology and Microbiology Dep., Texas A&M Univ., College Station TX
77843; P.F. Lamb and G.R. Carlson, Northern Agricultural Research
Center, 3848 Fort Cir., Havre, MT 59501. Received 20 Mar. 2009.
*Corresponding author: (usslt@montana.edu).
Abbreviations: CIM, composite interval mapping; DArT, diversity
arrays technology; LOD, logarithm of odds; QTL, quantitative trait
loci; RIL, recombinant inbred lines; SSR, simple sequence repeat;
WSS, wheat stem sawfly.
A
n initial step in insect herbivory is selection of the host plant
for feeding or oviposition. Lack of host attractiveness in certain genotypes to ovipositing females may provide a level of resistance to insect damage. Exploitation of this type of antixenotic
resistance in plant breeding requires that the genotypes of the host
plant show heritable variation in the number of eggs deposited by
females. Interaction of the wheat stem sawfly (WSS; Cephus cinctus
Nort.) with wheat (Triticum aestivum L. em. Thell.) provides a system to assay the genetics of host plant interaction with egg-laying
females for an agriculturally significant insect pest.
Wheat stem sawfly inflicts severe economic damage to winter
and spring wheat in the Northern Great Plains of the United States.
Infested plants produce lower yields and usually lodge after girdling
of the stem base by mature larvae, reducing the amount of grain
that can be harvested (Holmes, 1977; Morrill et al., 1992). Wheat
stem sawfly larvae overwinter in below-ground portions of stems,
Published in Crop Sci. 50:73–86 (2010).
doi: 10.2135/cropsci2009.03.0145
© Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
All rights reserved. No part of this periodical may be reproduced or transmitted in any
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the publisher. Permission for printing and for reprinting the material contained herein
has been obtained by the publisher.
CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
73
and the adults emerge in the early summer. Mating is brief,
and the females spend the balance of their lifespan seeking
large-stemmed grass hosts for oviposition (Ainslie, 1929;
Weiss and Morrill, 1992). Because adults are short lived,
rapid location and selection of hosts is critical for continuation of their life cycle.
Despite considerable effort to control WSS proliferation and migration by cultural, chemical, and biological
methods, only host plant resistance has proven to be both
reliable and effective. Host plant resistance is found in
wheat accessions that have stems fi lled with pith, referred
to as solid stems (Kemp, 1934). The pith impedes larval
growth and migration, greatly reducing stem cutting,
insect survival, and ultimately population growth (Wallace and McNeal, 1966). Genetic experiments have shown
that a single locus, termed Qss.msub-3BL, contributed at
least 76% of the total variation for stem solidness in a cross
between solid- and hollow-stemmed parents (Cook et al.,
2004). A second minor gene for stem solidness (Qssmsub3DL) was identified in a separate population (Lanning
et al., 2006). Resistance conferred by solid stems may
be due to a combination of antibiosis (death of neonates
and young larvae) in the stem and tolerance (inability of
mature larvae to cut the stem). However, solid stems do
not confer complete resistance, and cutting as high as 30%
may be observed occasionally in solid-stem wheat cultivars (Lamb and Carlson, unpublished data, 2008).
Another type of resistance, antixenosis, may be available for genetic control of the WSS (Weaver et al., 2009).
Antixenosis refers to reduced ability of plants to serve as a
host through mediation of insect behavior. Attraction of
phytophagous insects to volatile chemicals produced by
host plants has been shown in plant–herbivore systems.
Insects can use plant volatiles as cues to select appropriate hosts for feeding and oviposition (reviewed by Visser,
1986; Bernays and Chapman, 1994; Schoonhoven et al.,
1998; Bernays, 2001). Nonpreference for oviposition, a
form of antixenosis, serves as a resistance mechanism in
sorghum [Sorghum bicolor (L.) Moench.] to the sorghum
midge, Stenodiplosis sorghicola (Coquillet) (Henzell et al.,
1994). Tao et al. (2003) identified two quantitative trait
loci (QTL) explaining 12 and 15% of the variation for
nonpreference among 120 recombinant inbred lines (RIL)
in a sorghum cross.
We report here that WSS females are differentially
attracted to spring wheat cultivars for the purpose of oviposition, with Reeder being a preferred host, while Conan
is less preferred. We analyzed a recombinant inbred line
population derived from a cross between Reeder and
Conan spring wheat to determine the genetic basis for
differences in WSS attraction, and to identify molecular
markers that may be useful for breeding.
74
MATERIALS AND METHODS
Plant Materials
Ninety-one F6 –derived RIL were developed by single-seed
descent beginning at the F2 generation from a cross between
Reeder (PI 613586) and Conan (PI 607549) spring wheat.
Reeder is a semidwarf cultivar due to alleles at Rht-B1 and
Conan is semidwarf due to alleles at Rht-D1, and thus the progeny lines segregated for height. Semidwarf and standard-height
lines were retained for this experiment. The extreme dwarf phenotype conferred by reduced height alleles at both loci resulted
in poor vigor in low-moisture areas such as the sawfly sites used
for this study, and therefore were removed from the population. Dwarf RIL would also be relatively inaccessible for WSS
infestation due to very short stems in the Montana testing sites.
Agronomic and Sawfly Infestation Data
The RIL were planted in 2006 and 2007 at two locations in
northern Montana with a history of WSS infestation. Locations
included private farms north of Havre (48°49'55" N, 110°03'28"
W) and near Loring (48°46'31"N, 107°52'33" W). Experiments
were established in late April in the midst of stubble from a
previous WSS-infested wheat crop. Each experiment was a randomized block with three replications. Each block included the
91 RIL along with four plots of each parent. Plots consisted
of 10 seeds per entry planted in individual hills, with spacing
of 0.8 m between adjacent hills. For all four sites, stems were
individually collected at maturity in late August of each year.
Every stem was dissected to determine the presence of larvae
(infestation) and larval survival (stem cutting). Stem dissection
also revealed varying levels of parasitism by endemic natural
enemies (Runyon et al., 2002). Stem diameter was also measured at this time and the mean values for all of the stems in
each plot were used for statistical analysis.
Infestation and cutting made it impossible to accurately
record agronomic data at infested sites. Therefore, separate trials were established in Bozeman in 2006 and 2007 at the Arthur
Post Research Farm (45°41'N, 111°00'W) to obtain agronomic
data. These trials consisted of single row plots 3 m in length with
0.30 m between rows seeded at a rate of 2.3 g m–1. Each experiment was planted in a randomized block design with three replications in which each block included 91 RIL and four plots of
each parent. Plant height, excluding awns, was measured as mean
height of plants within a row. Heading date was measured as the
number of days from planting when 50% of the heads were completely emerged. The level of pith in each internode was measured
on a scale ranging from 1 to 5, where 1 was considered hollow and
5 was solid. Ratings at each of the five internodes were summed
to provide a total stem solidness score between 5 (hollow) and 25
(solid) for each stem. A mean value of five stems per plot was used
for statistical analysis. Glaucousness was visually assessed on a scale
of 1 (low) to 5 (high) for the two uppermost internodes and the
spike for each plot in the 2007 field trial. The three readings were
summed to give a final scale of 3 (low) to 15 (high).
All response variables were analyzed via analysis of variance
using a model for a randomized complete block for each environment. Subsequently the analysis was then combined over environments where the model included environment, replication within
environment, entry, and environment × entry using PROC
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CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
GLM in SAS (SAS Institute, 2004). All factors except environments were considered random effects. Narrow-sense heritability
for WSS infestation and cutting were computed on an entry mean
basis as described in Fehr (1987) for each environment and combined over locations. Pearson correlations were computed using
PROC CORR in SAS among WSS infestation, cutting, parasitism, and agronomic–maturity traits using entry mean values from
the hill plot experiments and the single row yield trial.
Map Construction
Genomic DNA was isolated from leaf tissue as described by
Riede and Anderson (1996).
Reeder and Conan were screened with about 700 simple
sequence repeat (SSR) markers listed on GrainGenes (http://
wheat.pw.usda.gov/cgi-bin/graingenes/browse.cgi
[verified
20 Oct. 2009]) to identify polymorphic markers. Approximately 50 ng of DNA was used in polymerase chain reaction as
described by Roder et al. (1998), with annealing temperatures
as suggested on GrainGenes. Polymorphic SSR markers were
used to assay the entire set of 91 RIL and 232 were mapped. To
increase the number of markers and combine linkage groups,
the population was also submitted for diversity arrays technology (DArT) analysis (Akbari et al., 2006; http://www.triticarte.
com.au/default.html [verified 20 Oct. 2009]). A total of 314
DArT markers were identified as polymorphic. However, many
of these cosegregated, so that a total of 190 DArT markers were
used in map construction. In addition to DArT and SSR markers, the RIL were screened with markers for known genes that
were polymorphic between Reeder and Conan, including PpdA1, Ppd-B1, Ppd-D1 for photoperiod sensitivity (Beales et al.,
2007), Rht-B1 and Rht-D1 for semidwarf growth habit (Ellis et
al., 2002), and VrnB1 (Zhang et al., 2008) for response to vernalization. High-molecular-weight glutenins were assayed following the method of Payne et al. (1981) except that di-thiothreitol
was used as a reducing agent rather than 2-mercaptoethanol.
Additionally, primer sets linked to low-molecular-weight glutenin/gliadin genes that differed between Reeder and Conan
were used to screen the population. These included psp1 for the
Glu-A3 locus and psp2 for the Gli-B1 locus (Devos et al., 1995).
The genetic map was constructed using MapMaker V3.0
(Lander et al., 1987; Lincoln et al., 1993). First, selected markers were anchored to chromosomes using information from the
wheat consensus map (Somers et al., 2004). The ASSIGN command was used to identify linkage groups with a logarithm of
odds (LOD) threshold of 3.0. Each group was ordered using the
THREE-POINT (at minimum LOD 3.00, maximum distance
37.2) and ORDER commands where possible. If an order could
not be determined in this fashion, then the SUGGEST SUBSET, COMPARE, TRY, and NEAR commands were used.
Where cosegregating DArT markers confounded ordering, all
but one was dropped from the framework to get an initial order.
Final marker orders were checked with the RIPPLE command
(window size: 5, log-likelihood threshold: 2.00). Recombination
distances were determined using the Kosambi mapping function
(Kosambi, 1944). The map was also built using MapDisto (http://
mapdisto.free.fr/ [verified 20 Oct. 2009]; Lorieux et al., 1995),
using the population identifier “single-seed descent.” The FIND
GROUPS command was used first (LOD 5 minimum and rmax
0.3). The LOD value was set high initially to create linkage
CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
groups of reasonable size. Each linkage group over five loci was
ordered using the ORDER SEQUENCE command, the Seriation II method (Buetow and Chakravarti, 1987a, 1987b). Groups
of fewer than five loci were ordered using COMPARE ALL
ORDERS. The RIPPLE command was used to check orders
using SARF criteria and progressive methods. Once orders were
determined, the data were searched for errors and 31 likely errors
were replaced with missing data. The DROP LOCUS command
was used, and two loci were dropped as their removal decreased
the size of the map. The recombination fraction estimate was
made using the classical command utilizing Martin and Hospital
(2006) (SSD). Recombination distances were determined using
the Kosambi mapping function (Kosambi, 1944).
Segregation Distortion
Markers were subjected to a chi-square test for fit to a 1:1 ratio
using MapDisto. Markers with significant distortion are indicated in the genetic map (Fig. 1).
QTL Mapping
QTL Cartographer (http://statgen.ncsu.edu/qtlcart/WQTLCart.htm [verified 20 Oct. 2009]; Wang et al., 2007) was used
to perform single marker analysis, interval mapping (Lander
and Botstein, 1989), and composite interval mapping (CIM)
(Zeng, 1993, 1994) for WSS infestation and cutting using the
entry means for each environment. The frequency distributions
for these traits were checked for normality for each location and
when combined over environments using the Shapiro–Wilk test
from PROC UNIVARIATE in SAS (SAS Institute, 2004).
Quantitative trait loci were identified and analyzed successively, first utilizing the simplest analysis to scan the whole
genome. Single marker analysis fit the data to the simple linear
regression model, giving the estimates for b0, b1, and the F statistic for each marker. For interval mapping, a walking speed
of 2 cM was used and significant LOD values were set by 300
permutations at an experiment-wise P < 0.05. Identified significant QTL were further analyzed with CIM. Cofactors were
determined using the standard CIM model, regression method
forward and backward with a probability in and out of 0.1. Composite interval mapping can identify linked multiple QTL and
help refine QTL peaks. Initially, LOD values were set by 300
permutations at an experiment-wise P < 0.05 and the window
size was 20. Significant QTL were further studied by narrowing
the window size to 5 and using 1,000 permutation tests to define
an empirical detection threshold at an experiment-wise P < 0.01
(Churchill and Doerge, 1994). Significant LOD values varied
between 2.5 and 3.5. The mean map position of the significant
QTL was established by the map position of the peak LOD score
in the interval between two flanking markers. A 1-LOD fall-off
(from the QTL peak) method was used to estimate the left-and
right-flanking map positions of a confidence interval surrounding the mean QTL map position (Chaky, 2003).
RESULTS
Agronomic and Sawfly Infestation Data
Wheat stem sawfly infestation occurred in all four test sites
(Table 1). The parental cultivars Conan and Reeder differed
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75
Figure 1. Conan–Reeder linkage map created using 91 F6 –derived recombinant inbred lines from single-seed descent (SSD) with a total
of 431 markers and a total map distance of 2608.72 cM. A total of 46 linkage groups were identified and associated with a chromosome
using the consensus map. Chromosome number is indicated in bold above each set of linkage groups, and sets of linkage groups were
ordered using the consensus map. To the left of the linkage groups is recorded the distance (in cM) between markers and to the right is
the marker name. The asterisk at the beginning of the marker name is generated by the mapping program. Asterisks at the end of the
marker name denote significantly distorted loci (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, *****P < 0.00001).
76
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Figure 1. Continued.
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Figure 1. Continued.
78
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significantly for both percent infestation and Table 1. Mean percent wheat stem sawfly infestation and cutting in 91 recombinant inbred lines (RIL) and parents in four replicated trials in Montana.
percent cutting at all sites. Percent infestation
Cultivar
RIL
averaged over all sites was 50.2% for Reeder
†
Location
Trait
Reeder
Conan
Mean
SD
Range‡ Heritability
and 15.9% for Conan. Percent cutting averInfestation
(%)
38.1
3.0
20.8
15.8
0.0–63.8
0.78
aged over all sites was lower than infestation Havre 2006
Cutting (%)
8.8
0.0
3.6
3.7
0.0–16.1
0.60
for both Reeder (34.3%) and Conan (10.8%).
Infestation
46.9
9.1
25.3
16.5
0.0–56.5
0.82
The RIL showed significant genetic varia- Loring 2006
Cutting
38.0
8.0
18.2
12.1
0.0–49.9
0.73
tion for both percent infestation and percent
Infestation
58.6
29.9
43.9
13.1
18.1–68.4
0.69
cutting (Table 1). The distribution of cutting Havre 2007
Cutting
46.3
20.1
34.3
11.0 12.1–61.4
0.70
and infestation among the RIL was not sigInfestation
56.8
21.2
48.6
15.1
13.7–77.3
0.34
nificantly different from normal for Havre Loring 2007
Cutting
43.9
14.6
33.6
10.4
4.5–72.4
0.36
2007, Loring 2007, or the average of all sites
Infestation
50.2
15.9
34.7
12.7
9.6–57.5
0.86
(Shapiro–Wilk ≥ 0.978572, P > 0.1342). Mean§
Cutting
34.3
10.8
22.4
7.5
6.1–47.5
0.75
However, the Havre 2006 and Loring 2006
†
Values
based
on
three
replications
of
four
hill
plots
per
replication
at
four
locations.
Differences
between
data were significantly different from norparents significant (P < 0.05) based on LSD.
mal (Shapiro–Wilk < 0.95718, P < 0.0042). ‡Values based on three replications for a single hill plot. Genotype effects were significant (P < 0.001) for all
The distributions of the Havre 2006 data trait/locations based on analysis of variance.
were skewed with less infestation and cut- §Mean values across environments and locations.
ting than expected for a normal distribuConan are both semidwarf, but Reeder at 83.9 cm is sigtion, possibly due to the braconid parasitoid as described
nificantly taller than Conan at 77.3 cm. Because Reeder
below. Genotype × environment interaction variance was
and Conan possess reduced height alleles at different loci,
also significant (P < 0.01) but small in comparison to genosemidwarf and standard-height genotypes were present in
type variance (data not shown). Narrow-sense heritability
the RIL population. The RIL exhibited heights of 70.5
values ranged from h2 = 0.34 (Loring 2007) to h2 = 0.78
to 111.8 cm. Reeder and Conan differed for the degree
(Havre 2006) for percent infestation, and from h2 = 0.36
of stem solidness, as Conan contains the major stem solid
(Loring 2007) to h2 = 0.73 (Loring 2006) for percent cutQTL identified by Cook et al. (2004). Conan had more
ting among the four sites. Heritability based on means over
solid stems than Reeder, 10.3 vs. 7.2, and solidness scores
environments was h2 = 0.86 for infestation and h2 = 0.75 for
differed significantly (P < 0.01) among the RIL. Reeder
cutting. Table 2 shows that the correlation between percent
and Conan also varied in glaucousness as measured visually
infestation and percent cutting was significant (P < 0.0001)
on a scale of 3 (low) to 15 (high). Conan received a score of
at all four sites. However, the correlation was lower at the
15 (± 0.11) and Reeder received a score of 4.9 (± 0.11). The
Havre 2006 site, reflecting the fact that most of the infested
RIL varied significantly for glaucousness (P < 0.05) though
stems were not cut by the WSS. One cause of sawfly mortalonly seven nonglaucous genotypes similar to Reeder were
ity that varies by geographic region is presence of endemic
braconid parasitoids (Runyon et al., 2002). Braconid paraTable 2. Correlation of percent wheat stem sawfly infestation
sitoids were evident in Havre 2006, with >50% of the larand percent cutting for 91 recombinant inbred lines grown in
vae parasitized in the stem by these native wasps. Parasitized
four locations in Montana.
larvae die before the stem is cut. The confounding effect of
Havre Loring Havre Loring Combined
Trait
the parasitoid is reflected in the relatively low correlation
2006 2006 2007 2007 locations
(r = 0.69) between infestation and cutting seen at this site.
Cutting/Infestation
0.69**** 0.95**** 0.96**** 0.87****
0.94****
The correlation between the sum of parasitism and cutting
Cutting plus
0.95**** 0.95**** 0.97**** 0.88****
0.97****
parasitism/infestation
to infestation was r = 0.95 at this site (Table 2), indicating
****
Probability of correlation different from zero is <0.0001.
that parasitism was the only obvious mortality factor preventing larvae from cutting stems. Parasitism was not a facTable 3. Mean agronomic data for Reeder, Conan, and 91
tor at any other location.
recombinant inbred lines (RIL) from single-row yield trials
The distant location of the WSS test sites as well as
grown in Bozeman, MT, in 2006 and 2007.
severe lodging due to sawfly damage prevented collection
Cultivar
RIL
of agronomic data at these sites. Thus, the RIL and parents
Trait
Reeder
Conan
Mean
Range
were evaluated in replicated trials for 2 yr at a site with
Heading date (d)†
60.2
60.7
61.3
56–68
no WSS infestation. Pertinent agronomic data for Reeder,
†‡
7.2
10.3
8.3
7.1–10.8
Stem solidness
Conan, and the RIL from these experiments are presented
83.9
77.3
92.3
70.5–111.8
Plant height (cm)†‡
in Table 3. Although heading date for Reeder and Conan
†
Genotype effects were significant (P < 0.001) for all trait/environments based on
was similar, the RIL showed transgressive segregation
analysis of variance.
‡
Differences between parents significant (P < 0.05) based on least significant diffor heading date, with a range of 56 to 68 d. Reeder and
ference.
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79
Table 4. Correlation of wheat stem sawfly infestation and cutting with maturity and agronomic traits for 91 recombinant
inbred lines from the Reeder–Conan cross. Data are based
on means over four locations for cutting and infestation and
means over two locations for heading date, plant height, and
stem solidness.
Trait
Heading date
Plant height Stem solidness
Sawfly cutting
–0.55***
0.028 ns†
–0.32**
Sawfly infestation
–0.57***
0.11 ns
–0.31**
**
Probability of correlation different from zero is <0.01.
***
†
Probability of correlation different from zero is <0.001.
ns, not significant.
identified. Conan and Reeder also varied significantly for
stem diameter, with Conan stems having a larger diameter
(3.5 ± 0.04 mm) than Reeder stems (3.1 ± 0.04 mm). The
RIL also varied significantly for stem diameter (P < 0.05).
Correlation analysis (Table 4) showed that heading date was negatively associated with WSS infestation
(r = −0.57) and cutting (r = −0.55), indicating that earlier
heading was associated with increased infestation and cutting. The fact that WSS data and agronomic data were
obtained in different locations possibly had some impact
on the observed correlations. However, the correlation of
line performance for heading date between the Bozeman
site and a non–sawfly testing site in Havre (48°54'32" N,
109°67'45" W), based on data from advanced breeding trials containing 64 lines, was r = 0.76 in 2006 and r = 0.65
in 2007 (Lanning and Talbert, unpublished data, 2006,
2007). The correlation of line performance between these
sites for plant height was r = 0.62 in 2006 and r = 0.86 in
2007. Thus, plant height and heading date measurements
from Bozeman were likely a reasonable reflection of performance in the north Havre testing site. Degree of stem
solidness was negatively correlated with percent cutting
(r = −0.32) and percent infestation (r = −0.31). No significant correlation was observed between glaucousness, plant
height, or stem diameter with either infestation or cutting.
Construction of Genetic Map
A genetic map with a total of 431 markers and a total size
of 2608.75 cM was created for the Reeder–Conan RIL
(Fig. 1). The map contained 232 SSR markers, 190 DArT
markers (Akbari et al., 2006), six markers for major genes
controlling development (Ppd-A1, Ppd-B1, Ppd-D1, Rht-B1,
Rht-D1, Vrn-B1), and three markers for storage proteins. A
total of 46 linkage groups were identified. Chromosomal
assignments of linkage groups were tentatively made by
referring to previous maps (Somers et al., 2004). Where
possible, the map order was compared with the consensus map and deletion maps (http://wheat.pw.usda.gov/
ggpages/SSRclub/GeneticPhysical/ [verified 20 Oct.
2009]). There was overall agreement between the order of
markers in this map and previously published maps. The
six markers for major genes and storage proteins all mapped
80
to the appropriate chromosomes (i.e., Ppd-A1, 2A; Ppd-B1,
2B; Ppd-D1, 2D; Rht-B1, 4B; Rht-D1, 4D; Vrn-B1, 5B; GluA3, 1A; Glu-B1 and Gli-B1, 1B) (Fig. 1). The distribution
of markers and linkage groups among the chromosomes is
shown in Fig. 1. In most cases, the D genome chromosomes
had fewer mapped markers within a homeologous chromosome group. Chromosome 5D is an exception, with
more markers than 5A. In several cases either few or no
DArT markers mapped to a D genome chromosome. The
homeologous group 6 chromosomes overall had the fewest
mapped markers.
Patterns of Segregation Distortion
Chi-square (χ2) analysis indicated 66 loci with segregation distortion where segregation was significantly different from the expected 1:1 ratio (Fig. 1). Approximately 20
of the distorted loci occurred singly, while the remainder
were distributed in clusters of distorted loci on linkage
groups 2D, 4A, 4B, 4D, 5B, 6B, 7B, and 7D. Clusters with
the most significant distortion were associated with the
Rht loci on 4B and 4D or were on 5B or 7D. Distorted loci
favored the Conan allele on 2D, 4A, 4B, 5B, 7B, and 7D,
while the Reeder allele was favored on 4D, 6B, and 7B.
QTL Analysis
Single marker analysis showed QTL for infestation and
cutting based on means across the four locations associated
with the Ppd-D1 locus. This locus also had an effect on
heading date, with insensitive genotypes being earlier and
having greater WSS infestation and cutting and has been
previously mapped to the short arm of 2D. Conan had the
insensitive allele at the Ppd-D1 locus, while Reeder had the
sensitive allele. Ppd-D1 was identified as influencing heading date by interval mapping (results not shown) and CIM
(Fig. 2). Single marker analysis also identified an important
QTL for infestation and cutting based on mean data over
four locations on chromosome 3B defined by SSR marker
gwm340. This marker identifies the solid stem locus Qss.
msub.3BL (Cook et al., 2004), which was associated with
QTL for cutting and infestation in one environment as
detected through interval mapping (results not shown) and
CIM (Fig. 2). The plot for chromosome 3B is discontinuous because it consisted of two linkage groups. Quantitative trait loci associated with infestation or cutting in fewer
environments were identified on chromosomes 1B and 5B
(Table 5). These QTL were also associated with heading
date. The QTL on 5B was associated with VrnB1.
Using single marker analysis, markers on chromosomes 1B, 2D, and 4A, which did not cosegregate with
morphological traits, showed significant association
(P < 0.05) to infestation in all four locations. Of these,
interval mapping indicated significant QTL on chromosomes 2D and 4A (results not shown). Interval mapping
produced a broad curve that involved multiple markers
WWW.CROPS.ORG
CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
Figure 2. Chromosome maps (x axis) and logarithm of
odds (LOD) scores (y axis) for chromosomes 2D, 3B,
and 4A generated through composite interval mapping.
Several marker designations and relative positions are on
the x axis. Refer to Fig. 1 for complete maps. The LOD
graph indicates strength of association between markers
and trait as follows: heading date—hd67: combined 2006
and 2007 data from both locations; solid stem—ssolid67:
combined 2006 and 2007 data from both locations;
infestation—I67: combined 2006 and 2007 data from both
locations, IH7: Havre 2007, IL7: Loring 2007, IL6: Loring
2006, cutting—C67: combined 2006 and 2007 data from
both locations, CH7: Havre 2007, CL7: Loring 2007, CL6:
Loring 2006. Horizontal line indicates significant LOD
scores. The lower graph indicates the additive effect of
the Reeder allele.
including about 50 cM on 2D and about 20 cM
on 4A. Composite interval mapping localized
the QTL to narrower intervals, as seen in Fig.
2. The 2D QTL was significant for infestation
and approached significance for cutting based on
the mean of all four environments, as well as cutting for Havre 2007 and cutting and infestation
Loring 2006. Composite interval mapping discriminated two QTLs mapping to 4A that had
opposite effects (Fig. 2). For the QTL between
markers gwm494 and wmc760, the Reeder allele
increased cutting and infestation, while with the
QTL occurring between wmc760 and barc243
the Reeder allele decreased infestation (Fig. 2).
The QTL between wmc760 and barc243 was not
significant until cofactors correcting for the effect
of the other QTL were used. Traits significant at
these QTL included cutting based on the mean
of all four environments, as well as the cutting
and infestation for Loring and Havre 2007. The
QTL on chromosome 2D explained as much as
19% of the variation for mean infestation and cutting. The QTL on 4A for which Reeder contributed the allele for increased cutting and infestation
explained as much as 29% of the variation while the
linked QTL explained as much as 15%. The QTL
on chromosomes 2D and 4A did not cosegregate
with markers showing segregation distortion or
with QTL for morphological and developmental
traits such as heading date or stem solidness.
DISCUSSION
Previous small-plot studies indicated that spring
wheat cultivars differed in their attractiveness to
egg-laying females of WSS. Through these assessments of stem lodging for several location/years,
Conan and Reeder were identified as extremes
in levels of attractiveness to WSS (Weaver et al.,
CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
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81
Table 5. Significant quantitative trait loci (QTL) identified through composite interval mapping for stem cutting or infestation by
the wheat stem sawfly at four locations† in Montana.
Chromosome
Flanking markers
Location/trait
R2‡
LOD§
Effect of Reeder allele
Coincident QTL
cfd20-cfd48
Havre06/infestation
0.08
2.5
Earlier heading, more cutting
Heading date
Ppd-D1-barc168
Loring07/cutting
Combined/cutting¶
0.12
0.10
4.8
3.0
Later heading, less cutting
and infestation
Heading date
gwm539-cfd44
Loring06/infestation
Loring06/cutting
Havre06/infestation
Havre06/cutting
Havre07/infestation
Havre07/cutting
Combined/cutting
Combined/infestation#
0.10
0.19
0.10
0.10
0.13
0.15
0.10
0.16
2.6
5.0
3.2
3.3
3.5
4.2
2.4
3.8
More cutting and infestation
None
gwm299-barc77
Loring06/cutting
Loring06/infest
0.09
0.14
3.5
3.2
Decreased solidness, more
cutting and infestation
Stem solidness
barc164-gwm566
Loring07/cutting
0.10
4.0
Decreased cutting
None
wmc760-wPt1743
Loring07/infestation
Havre07/infestation
Havre07/cutting
Combined/cutting¶
0.07
0.15
0.14
0.11
3.0
4.6
5.6
4.0
Decreased cutting and infestation
None
gwm494-wmc760
Loring07/infestation
Havre07/infestation
Loring07/cutting
Havre07/cutting
Combined/cutting¶
0.29
0.29
0.20
0.27
0.2
6.0
6.0
6.5
7.0
6.0
More cutting and infestation
None
Havre06/infestation
0.26
4.0
Earlier heading, more cutting
and infestation
Heading date
1B
2D
3B
4A
5B
†
barc74-gwm604
Locations: Havre06, Havre 2006; Havre07, Havre 2007; Loring06, Loring 2006; Loring07, Loring 2007.
‡
The phenotypic variation explained by the QTL.
§
LOD, logarithm of odds.
¶
Average of cutting over all sites.
#
Average of infestation over all sites.
2009). Similar behavior of WSS was seen in the Reeder
and Conan hill plots of the current study, as WSS females
in all four locations infested more Reeder stems than
Conan. The ability of female sawfl ies to distinguish among
genotypes proved to be highly heritable (Table 1). The
lower heritability value at the Loring 2007 site may have
been due to an infestation of wild oats (Avena fatua L.) that
encompassed about one-half of each replication. Wild oats,
a host for the WSS, may have confounded WSS oviposition
preference among the RIL, as described by Sing (2002).
Given the complexities of insect reproductive behavior
(Bernays and Chapman, 1994), the WSS was remarkably
consistent in selection of host plants for egg laying. These
consistent results for parental infestation levels are reflected
in the high heritability values for this trait.
Percent cutting was lower than percent infestation in
all four experiments. This is expected, given that no cutting occurs when larvae die before moving to the base of
the stem. One cause of WSS mortality that varies by geographic region is the presence of endemic braconid parasitoids (Runyon et al., 2002). In fact, these parasitoids were
present in high numbers in the Havre 2006 site and caused
a high level of mortality of WSS. The effect of the parasitoids on WSS mortality is reflected in the relatively low
82
correlation (r = 0.69) between infestation and cutting seen
in this site compared with other sites. The distribution of
the data from this site was also skewed, probably due to
the parasitoid. The correlation between percent infestation
and the sum of percent cutting and percent parasitism was
r = 0.95, suggesting that the low level of cutting in this
environment was due to parasitism.
Oviposition preference in WSS has been reported to
be influenced by developmental stage and the degree of
stem solidness (Holmes and Peterson, 1960). Adult WSS
females are constrained to stems that they can breach with
their sawlike ovipositor and are thus dependent on the
brief availability of rapidly growing, soft green shoots.
In wheat, stems that have begun elongating and develop
through to the full emergence of the head are preferred.
Among suitable stems in several host species, females select
the taller ones for oviposition (Seamans, 1928; Holmes
and Peterson, 1960; Youtie and Johnson, 1988; Morrill et
al., 1992; Perez-Mendoza et al., 2006). Wheat stem sawfly
females are also limited by their body size in the range of
stem diameters that they can infest because they encircle
the stems with their legs when ovipositing, so there is a
variable range of stem diameters that is utilized among
host species (Youtie and Johnson, 1988; Perez-Mendoza
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CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
et al., 2006). Morphological traits that did not cosegregate
with oviposition in the RIL were stem diameter and glaucousness. Stem diameters for Conan and Reeder and the
RIL were all within the usable range of the WSS (Morrill
et al., 1992).
The Conan–Reeder RIL were segregating for morphological characteristics that appeared to influence WSS
oviposition and cutting, including stem solidness and heading date. The RIL showed greater variation for heading
date and plant height than the variation between Reeder
and Conan. Height variation among the RIL is attributable to the fact that Reeder and Conan contained different
genes for semidwarf growth habit, resulting in segregation in the RIL. Semidwarf and standard-height genotypes
were present among the RIL. Similarly, the range of variation for heading date in the RIL suggests that Reeder and
Conan differ for genes influencing this trait as well. Previous work has shown that the insensitive allele at Ppd-D1
confers earlier heading and shorter stature in the Northern
Great Plains (Dyck et al., 2004). While Conan and Reeder
are both photoperiod insensitive, Conan has the Ppd-D1
allele conferring photoperiod insensitivity and Reeder had
the sensitive allele at the same locus. Reeder has an insensitive allele at Ppd-B1, while Conan has the sensitive allele at
this locus (Blake et al., 2009).
The negative correlations between heading date and
WSS infestation (r = −0.57) and cutting (r = −0.55) has
important implications for breeding WSS-resistant cultivars. Early lines had stems available for oviposition by the
WSS for a longer period of time, and were susceptible
to egg laying during early stages of sawfly fl ight in the
spring. Thus, selection for later heading lines may help
control the WSS (Morrill and Kushnak, 1999). However,
earlier heading has been an important selection target for
hard red spring wheat in the Great Plains, in that early
lines have the ability to head and begin to fi ll grain before
extreme summer heat. Thus, breeding objectives for sawfly resistance and heat resistance may be in opposition to
one another, emphasizing the need for the identification
of novel mechanisms of resistance.
Height did not significantly correlate with infestation
or cutting in a consistent manner in this population. Relationships between height and suitability of stems for oviposition typically include a broader range of height than
were observed in this study (Seamans, 1928, Holmes and
Peterson, 1960). We also did not observe significant QTL
for cutting or infestation associated with either of the Rht
genes segregating in this population, probably due to the
removal of the dwarf genotypes from the population.
The degree of stem solidness was correlated with
both WSS infestation (r = −0.31) and cutting (r = −0.32).
This result has been shown previously and, in fact, solid
stems are the primary plant characteristic used for WSS
management. Conan is referred to as a semisolid cultivar.
CROP SCIENCE, VOL. 50, JANUARY– FEBRUARY 2010
On a scale of 5 to 25, where 5 is hollow and 25 is completely pith-fi lled or solid, Conan averaged 10.3 vs. 7.2 for
Reeder (Table 3). The degree of solidness in either parent
is not sufficient to provide resistance to the WSS. The
primary resistant cultivar grown in Montana WSS areas
is Choteau (Lanning et al., 2004), with an average stem
solidness rating of 21.2. Even with a comparatively lower
level of stem solidness, it is interesting to note that solidness QTL could be identified in the Conan–Reeder RIL
as influencing infestation and damage caused by the WSS.
Current strategies for developing cultivars resistant
to WSS involve measuring stem solidness and selecting
genotypes with low stem cutting when breeding populations are evaluated in naturally infested areas. Our results
show that selection for low stem cutting may indirectly
select for later heading types as well as for stem solidness.
Delayed heading is impractical for agronomic reasons,
and solid stems are already used as a resistance mechanism
(Lanning et al., 2004). Thus, we were interested in identifying novel loci that influenced oviposition preference of
the sawfly without influencing agronomic performance.
An initial step was construction of a linkage map
with the Reeder–Conan RIL. We found that approximately 30% of SSR markers were polymorphic between
Reeder and Conan, and could be mapped using the RIL.
The DArT analysis identified 314 polymorphic markers
between the parents. Many of these mapped to the same
locations, leaving a total of 190 that were useful for complementing the SSR map. Chromosomes averaged 19.7
markers ranging from 37 on 1A and 3B to 7 on 1D and 6D
with most of the D genome chromosomes having less than
the average. Others also have reported a paucity of markers on the D genome, suggesting a need for additional
marker development (Francki et al., 2009). We identified a
total of 46 linkage groups instead of 21, indicating gaps on
some chromosomes due to lack of polymorphism between
Reeder and Conan. The presence of chromosome regions
that lack polymorphism between Reeder and Conan was
not unexpected, in that both are hard red spring wheat
cultivars adapted to the Northern Great Plains, and likely
share common ancestors. Pedigrees of the cultivars cannot be directly compared, in that Conan’s parents derived
from a recurrent selection population involving multiple parental lines (D. Clark, personal communication,
2007). Although the size of this map is comparable to
other wheat maps (current map—2608.75 cM; consensus
map—2569 cM) (Somers et al., 2004), the current map is
larger than it appears because all chromosome segments
were not linked. It is important to emphasize that linkage
groups containing few markers are tentatively assigned to
chromosomes because several markers have been previously mapped to more than one chromosome. The most
likely assignment was made by choosing the chromosome
to which most of the markers were previously mapped.
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83
At least one marker showing segregation distortion
was observed on every chromosome except 1D. Segregation distortion manifests in this population as both single
markers scattered through the genome and as groups of
distorted loci observed on several chromosomes Segregation distortion can occur for a variety of reasons (see Song
et al. [2006] for review). Some of the observed distortions
in this case may be due to population structure, as RIL
populations tend to have more distortion than F2 populations (Wang et al., 2003). The markers surrounding the Rht
genes were distorted because of our elimination of dwarf
lines from the population. The alleles conferring standard
height were favored in both cases. Previously, 1D, 3D, 4D,
5B, and 7D have been reported to carry segregation distorted loci in wheat and wheat relatives (Faris et al., 1998;
Kumar et al., 2007). We too observed segregation distortion on 4D, 5B, and 7D. While groups of distorted loci that
favored both parents were observed, most of the distorted
loci favored the Conan allele. Map distances corrected for
segregation distortion using the Bailey function (Bailey,
1949; Lorieux et al., 1995) for our population retained
marker order but increased distances between markers
beyond that expected based on previous maps (data not
shown). The classical function better represented expected
map distances and enabled the inclusion of cosegregating
loci that could not be mapped using the Bailey function.
Xu (2008) showed that the effect of segregation distortion
on QTL detection is likely to be negligible, though slight
positive or negative effects are possible. Negative effects
may include a slight loss in power of detection. Our inability to identify a QTL for cutting or infestation at the Rht
loci may be due to this loss of power. However, the novel
WSS infestation/cutting QTL on 2D and 4A were not in
regions showing segregation distortion.
Potential QTL for antixenosis that would decrease
WSS attraction without implications on agronomic performance were identified as QTL that did not cosegregate
with heading date and stem solidness. Also, QTL for WSS
infestation and cutting present in multiple environments
were of most interest. Quantitative trait loci on chromosomes 2D and 4A fit the criteria of no association with
morphological or developmental traits and having an effect
in multiple environments. Generation of additional markers for these regions is an important priority for eventual
manipulation of the QTL using marker-assisted selection.
Previous results have suggested that differential production of volatile compounds may control attraction of
insects to specific genotypes (Reddy et al., 2004; Martel
et al., 2005). Piesik et al. (2008) showed that wheat stems
reliably produce several volatile compounds that influence attraction of female WSS for oviposition. Reeder
and Conan differ for the production of these compounds
(Weaver et al. (2009). Markers identified in this study will
allow derivation of near-isogenic lines to determine the
84
genetic relationship between volatile production and the
newly identified QTL.
Two strategies for the use of QTL influencing antixenosis
can be envisioned for the control of WSS. First, QTL decreasing attractiveness could be backcrossed into high-yielding
cultivars to decrease infestation. Second, a solid-stemmed
cultivar with high attractiveness to female sawflies may prove
useful as a trap crop for growers wishing to expand the range
of available cultivars (Shelton and Badenes-Perez, 2006). The
present study is an initial step toward the development of the
necessary genetic tools for either approach. Our findings suggest that genetic manipulation of host preference may be used
to influence infestation in wheat as a complement to the host
plant resistance conferred by solid stems.
Acknowledgments
This research was partially supported from funding from
USDA-CSREES-NRI-CAP
award
2006-55606-16629,
USDA-Western SARE award SW07-025, USDA Special Grants
entitled “Novel semiochemical- and pathogen-based management strategies for wheat stem sawfly,” the Montana Wheat and
Barley Committee, and the Montana Board of Research and
Commercialization Technology.
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