Midge 2011 ADF20070092 Final Report · 2018-10-23 · 3 Treatments were 4 resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties

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2011 Final Report to ADF Project number: 20070092 Title: Agronomic and quality impacts of midge (Sitodiplosis mosellana) on wheat Authors: DePauw, R.M.2, Fenn, D.3, Fox, S.3, Lukow, O.3, Procunier, D.3, Smith, M.3, Vera, C.L.1

and Wise, I.3

1AAFC, MRF, Melfort, PO Box 1240, Melfort, SK, S0E 1A0 2AAFC, SPARC, PO Box 1030, Swift Current, SK, S9H 3X2 3AAFC, CRC, 195 Dafoe Rd, Winnipeg, MB, R3T 2M9 Introduction The wheat midge (Sitodiplosis mosellana) is an insect that has caused significant damage to wheat crops in Western Canada in recent years and was a major down-grading factor in 2006 and 2007. Damage is produced when a midge larva feeds on a developing wheat kernel, causing it to shrivel, crack and become deformed. Loss of shriveled kernels at harvest reduces yield, whereas retained damaged kernels can lead to reduction in grade and end-use quality performance. It has been estimated that midge caused about $40 million in losses in the 2006 Western Canadian CWRS crop ($19 million in grain yield loss and $21 million in down-grading losses). Midge populations are now established in central Alberta and producers are applying insecticide to provide some protection. A single antibiotic resistance gene (Sm1) was identified in soft winter wheat and transferred into spring wheat by breeders at several research establishments in western Canada. The Sm1 gene is located on chromosome 2BS; molecular markers have been developed for this gene; the gene has high penetrance, allowing for effective selection in the field when wheat midge are present (Thomas et al. 2005). As resistance based on a single gene is often short-lived, these resistant wheat genotypes have been proposed to be sold as the major component in varietal blends which will also include a small proportion of a susceptible cultivar, known as a refuge.

A refuge has been proposed to manage the expected mutations to virulence in wheat midge progeny. Since adult wheat midge tend not to move far from their sites of emergence prior to mating, it is important to provide an interspersed refuge so that sufficient numbers of avirulent midge surround any virulent midge that may emerge. Therefore, any virulent midge will most likely mate with avirulent midge, discouraging the retention of the mutation to virulence in the midge population. Providing a separate susceptible refuge would be inappropriate to manage mutations to virulence in the wheat midge as any virulent wheat midge that does emerge would be proximal to other virulent midge. A separate susceptible refuge would actually encourage survival of mutations to virulence in the wheat midge. Thus, the interspersed refuge is designed to ensure that sufficient numbers of avirulent adult midge are produced and secure that any virulent midge that may emerge will likely mate with an avirulent midge: this will delay the expected mutations to virulence from establishing in the midge population.

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Five wheat varieties have been registered recently (2008 and 2009), each possessing the Sm1 gene that confers resistance to wheat midge: Unity (BW362), Fieldstar (BW365), Shaw (BW394) and Goodeve (BW841) CWRS, and Glencross (ES95) CWES. These varieties will be marketed as varietal blends (ratio of 9 tolerant to1 susceptible). The susceptible components are: Waskada, in both Unity and Fieldstar, BA51*C222 in Shaw, AC Intrepid in Goodeve and Burnside in Glencross. The first seed sales are expected in the fall of 2009.

Varietal blends have not been used for certified seed sales of wheat in Canada. Producers, seed companies and regulatory agencies, including the Canadian Seed Growers Association and the Canadian Food Inspection Agency, are in need of information that details the stability of varietal blends over time. Many producers use farm-saved seed to produce wheat crops. Varietal blend stability will be necessary for producers to assess the economic impact of using their own seed and to protect the long term utility of the Sm1 midge resistance gene. Objectives

a) Mimic seed production and resistance management practices, including farm-saved seed production to assess impact on varietal blend stability over generations of production and potential loss of midge resistance. Evaluate the change in frequency of plants with and without the midge resistance gene Sm1 in varietal blends grown in environments with and without the presence of wheat midge.

b) To determine the agronomic value of midge resistance and protection of market grade. c) To study the impact of the presence of midge resistance on end-use quality.

These objectives will be addressed by two experiments at 8 locations:

(i) Effect of Sm1 gene on Agronomics and Grade Protection, using newly reconstituted varietal blends every year (AGP Midge Trial)

(ii) Genetic Drift by using Saved Seed (DSS Midge Trial)

Materials and Methods The study started in 2007 by establishing the AGP midge trial at 8 locations in the three prairie provinces: [Brandon (BRA), in Manitoba; Melfort (MEL), Indian Head (INH), Regina (REG), Saskatoon (SRC) and Swift Current (SWC), in Saskatchewan; and Lethbridge (LET) and Lacombe (LAC), in Alberta]. In 2008, and in subsequent years, the DSS midge trial was also established at each location, using seed harvested from each corresponding location. Plots in Lethbridge were irrigated.

The experimental design used was Randomized Complete Block (RCBD), with 4 and 5 replications for AGP and DSS trials, respectively.

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Treatments were 4 resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid).

Agronomic parameters measured were plant height, heading, maturity, lodging and seed yield. Seed was cleaned by running the seed through a white capper, to detach glumes (lemma and palea) still attached to the seeds, and subsequently through a dockage tester, to clean debris and smaller seeds, as per diagrams (Fig. 1).

Fig. 1. Seed cleaning procedure for the AGP midge trial

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Heads (20) were collected near maturity from the AGP trial (all plot and locations), to assess midge damage before harvest. The following parameters were determined or estimated from these samples: seeds per spike, seed weight, proportion of seeds damaged, proportion of harvestable seeds, proportion of non harvestable seeds (that could be potentially lost at harvest) and proportion of yield lost.

After the harvested seed was cleaned using a dockage tester, seed samples were sent to the

Canadian Grain Commission and from there to the Cereal Research Centre (CRC) for midge damage assessment. Harvested cleaned seed was also sent to the CRC for quality analysis, where the following parameters were measured: Minolta seed color, kernel weight, kernel size and kernel hardness.

Data was first analyzed using SAS procedure GLM to determine location and location x

treatment effects. Data was also analyzed using SAS procedure Mixed, having location as a random effect for final comparison among treatments, across locations.

Fig. 2. Seed cleaning procedure for the DSS midge trial

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Results Years 1 (2007), 2 (2008), 3 (2009) and 4 (2010) of the agronomic trials (AGP) and the genetic drift tests (DSS) have been completed for all locations. Genetic drift tests for 2010 are being completed. 1. Agronomic performance of midge resistant variety blends and susceptible varieties

GLM (SAS) analysis of 2010 data indicated that Treatment, Location and the interaction of these two factors were significant for most agronomic parameters measured.

Plant height was measured at all locations in 2010 (Appendix A, Fig. 3), with higher values

obtained at Saskatoon, Melfort, Lethbridge and Indian Head, and lower values at Swift Current, Brandon, Lacombe and Regina. Lower plants are usually associated with lower precipitation. Averaged across locations, the varietal blends BW394 VB and BW365 VB, and the susceptible varieties Katepwa and Waskada were taller, while the varietal blend BW362 VB and BW841 VB, and the susceptible varieties CDC Teal and AC intrepid were shorter (Fig. 3). Differences among varieties are usually genetic, showing a similar trend from site to site and from year to year.

Fig. 3. Plant height of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) from 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

90

92

94

96

98

100

102

104

106

108

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

Pla

nt

hei

gh

t (c

m)

6

Heading was recorded at six locations this year (Appendix A, Fig. 4). Plants at Saskatoon took less time (48.8 days from seeding) to start heading, which was the result of delayed seeding (13 June) at this location. Although the varieties did not show same heading pattern in all locations, AC Intrepid was consistently earlier heading in most locations where heading was measured (Fig. 4). This was also the case in previous years.

Maturity was measured at seven locations in 2010 (Appendix A, Fig. 5). Plants at Lethbridge took longest to mature, while at Saskatoon took the least. As with heading, there seemed to be a connection between maturity and seeding dates, but other factors, such as growing degree days (GDD) and soil moisture may play a stronger role in determining the length of this parameter. Averaged across locations, BW841 VB, Katepwa and AC Intrepid were the earliest maturing varieties, while BW362 VB, BW365 VB and Waskada were the latest (Fig. 5).

Lodging was reported at only four locations in 2010 (Appendix A, Fig. 6). Lodging was less

severe this year, with highest values recorded at Lethbridge. BW394 VB and BW841 VB and Intrepid showed lowest values, and BW 362 VB, BW365 VB and Katepwa the highest. Similar trend was observed across all locations (Fig. 6).

Fig. 4. Heading for 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) from 6 of 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

45

50

55

60

65

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

Hea

din

g (

day

s af

ter

seed

ing

)

7

Fig. 5. Maturity for 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) from 7 of 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

Fig. 6. Lodging for 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) from 4 of 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

100

102

104

106

108

110

112

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

Mat

uri

ty (

day

s fr

om

see

din

g)

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

Lo

dg

ing

(1-

9)

8

Seed yield was reported at all locations (Appendix A, Fig. 7), with highest values at

Lacombe (7322 kg ha-1) and lowest at Regina (2124 kg ha-1). The significant Location x Treatment interaction indicated that varieties did not behave similarly at all locations, but in general the resistant varieties BW362 VB and BW394 VB had the highest seed yields (Fig. 7). Same trend were observed in previous years. Percent dockage was measured at all locations (Appendix A, Fig. 8). In general dockage was low this year, with values <3% and in most cases <1.5%. Dockage may be somewhat affected by seed size (Appendix B, Fig. 9). This was observed in the seed from Swift Current in 2007, which were very small, compared to all other locations. The significant Location x Treatment interaction for this parameter indicated that the different varieties did not have consistent pattern across locations, however the resistant variety blend BW365 VB showed higher dockage values in most locations this year (Fig. 8), which also took place in 2007, 2008 and 2009. This variety also had the smallest seeds this year (Fig. 9), as well as in previous years.

Fig. 7. Seed yield for 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) from 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

3000

4000

5000

6000

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

See

d y

ield

(kg

ha

-1)

9

Fig. 8. Percent dockage of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) at 8 locations in the prairie provinces of Canada in 2009. Bars represent LSD values

Fig. 9. Seed size of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB and BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) at 8 locations in the prairie provinces of Canada in 2010. Bars represent LSD values.

20

25

30

35

40

BW362 VB

BW365 VB

BW394 VB

BW841 VB

Katepwa

CDC Teal

Waskada

AC Intrepid

Seed weight (g 1000-1)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

BW36

2 VB

BW36

5 VB

BW39

4 VB

BW84

1 VB

Katep

wa

CDC Tea

l

Wask

ada

AC Intre

pid

Do

ckag

e (%

)

10

2. Assessment of midge damage Objectives and Data Collected The objectives were: a) Assess consistency of two sampling methods (dissected spikes and harvested, cleaned seed from the Canadian Grain Commission, CGC) for percentage of midge damage in seed. b) Determine variation among wheat varieties and resistance class (resistant varietal blend or susceptible) in percentage of midge damage assessed by dissecting spikes and by examining harvested and cleaned samples from CGC. c) Determine effects of midge damage on seed weights: variation among wheat varieties and between resistance classes.

The experiment was set up as a Randomized Complete Block (RBC) design with four blocks (replicates) at each of eight locations. The same eight entries were used at each location: four were wheat-midge-resistant varietal blends (90% resistant with a 10% susceptible refuge), and four were susceptible cultivars. In the experimental design, entry was the fixed effect, and location and replicate were random effects. The locations selected were meant to be representative areas where midge populations and subsequent damage were expected to vary from virtually zero (likely southern Alberta) to high (likely east central Saskatchewan). The following data were collected from each plot at each location: a) dissected spikes: - number of undamaged (= not midge damaged) seeds - number of harvestable midge-damaged seeds - number of unharvestable midge-damaged seeds - 1000 kernel weight of undamaged seed - 1000 kernel weight of midge-damaged harvestable seed b) samples from Canadian Grain Commission (CGC): - percentage of midge-damaged seeds as assessed by CGC - number of midge-damaged seeds per 1000 undamaged seeds (assessed by Ian Wise) - 1000 kernel weight of undamaged seed - 1000 kernel weight of midge-damaged seed

Data were analyzed using the procedures of SAS. Midge-damage data were transformed when necessary to stabilize variances. Differences among means were considered significant at P < 0.05. Spikes for dissection from Lacombe contained only 6 midge-damaged seeds in total. Therefore, spike data from Lacombe were not included in analysis of midge damage. Analysis and Results a) Overall comparison among 2007, 2008, 2009 and 2010 midge damage levels Midge damage levels in 2010 were higher than in 2008 and 2009, but lower than in 2007. Except for BW394 VB (0.2-0.3% damage), percentage of midge-damaged seed among harvestable seed from dissected spikes ranged from 1.5% (Waskada) to 5.0% (CDC Teal), whereas damage

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assessments of harvested, cleaned seed by CGC personnel ranged from 1.0% (Waskada) to 2.8% (CDC Teal).

In 2009, percentage of damage among harvestable seed from dissected spikes ranged from 0.42% (BW394 VB) to 1.8% (BW365 VB). Assessments by CGC personnel ranged from 0.17% (BW394 VB) to 1.04% (CDC Teal). These ranges of midge damage were similar to those from 2008, when percentage of damage in dissected spikes ranged from 0.05% (BW394 VB) to 1.70% (CDC Teal), and in harvested, cleaned seed assessed by CGC personnel ranged from 0.07% (BW394) to 1.06% (CDC Teal). By comparison, 2007 midge-damage levels in harvestable seed from dissected spikes ranged from 3.72% (BW394) to 13.21% (BW365), and assessments by CGC personnel ranged from 1.87% (BW394) to 4.95% (AC Intrepid).

In 2010, as calculated from seed in dissected spikes, 9-40% of damaged seed in varietal

blends and 57-71% of damaged seed in susceptible varieties was considered unharvestable. Very similar results were obtained in 2007, 2008 and 2009. b) Consistency of two methods of sampling for percentage of midge damage

The two methods were done on independent samples of seed from the same plot. Data were analyzed as a split-plot analysis of variance (PROC MIXED), with sampling method as the subplot factor for each plot in the RCB design. Entry and sampling method were fixed effects; location, replicate within location and entry x replicate within location were random effects.

Seed samples from dissected spikes had overall damage levels that were slightly lower than actual damage (assessed by Ian Wise) in seed from CGC samples (P = 0.0317). There was also an interaction with entry (P = 0.0147), with the proportion of damaged seed in harvestable seed from dissected spikes being higher than in CGC samples for BW394 VB and Waskada (Figure 1). This suggests that, for most entries, a few small seeds from dissected spikes may have been incorrectly categorized as unharvestable.

These results contrast with the conclusion reached from analysis of the 2007, 2008 and 2009 data, that protocols used to separate damaged harvestable seed from damaged unharvestable seed from dissected spikes were not biased. c) Variation among wheat varieties in proportion of midge damage The proportion of midge-damaged seed was assessed at several levels: a) among all seed from dissected spikes b) among harvestable seed from dissected spikes, with unharvestable seed that would be lost in combining excluded from the samples c) among harvested, cleaned seed obtained from CGC; these samples were assessed by CGC personnel, and then by Ian Wise

Data were analyzed as a randomized complete block design, using a mixed model analysis of variance (PROC MIXED). Entry was a fixed effect; location and replicate within location were

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random effects. If the fixed effect was significant, comparisons among least-squares means were made using the Tukey-Kramer adjustment to separate means.

To compare proportions of midge-damaged seed between the resistant varietal blends and susceptible cultivars, the above analysis was repeated, with entry nested in resistance class (varietal blend and susceptible).

When all damaged seed was included (harvestable plus unharvestable), there were significant differences among entries in the proportion of damaged seed. Overall, susceptible cultivars had higher levels of damage than varietal blends, although damage to Waskada did not differ significantly from that of the varietal blends except for BW394 VB (Figure 2). Katepwa, CDC Teal and AC Intrepid had higher proportions of damage than Waskada, BW362 VB and BW841 VB. BW394 VB had less damage than any of the other entries. When only harvestable seed was included, CDC Teal and BW365 VB had higher proportions of damage than Waskada and BW394 (Figure 2). No overall difference was detected between susceptible cultivars and varietal blends.

Proportions of damaged seed in the CGC samples differed among entries as assessed by Ian Wise, who used a microscope to find all damaged seeds, and by CGC personnel. There were significant differences between assessment methods and between susceptible cultivars and varietal blends, but there was also a significant interaction between these two factors. For susceptible cultivars, actual midge damage levels as assessed by Ian Wise were 49% higher than those assessed by CGC personnel. For varietal blends, the actual midge damage levels were an average of 42% higher than assessments by CGC personnel. d) 1000 kernel weights of midge-damaged seed from dissected spikes and CGC samples

Some of the variation in weights of midge-damaged seed among entries can be explained by variation in the expected weight of the seed if it was not damaged. To control for this source of variation, weight of undamaged seed from the same sample was used as a covariate to adjust damaged seed weights. Analysis of covariance was carried out (PROC MIXED) on seed weight data from dissected spikes and from CGC samples. When weights of harvestable midge-damaged seed from dissected spikes were adjusted for the covariate, there were no significant differences among entries. (Figure 4). When weights of midge-damaged seed from cleaned CGC samples were adjusted for the covariate, there were differences among entries. Comparisons of adjusted least squares means showed that CDC Teal, AC Intrepid and Katepwa had larger damaged seed than BW841 VB and BW365 VB, relative to the undamaged seed (Figure 5).

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Figure 1. Proportion of midge-damaged seed in harvestable seed from dissected spikes and in seed from harvested, cleaned samples used for CGC assessments (actual damage assessed by Ian Wise) in 2010.

Figure 2. Proportion of midge-damaged seed in dissected spikes, 2010: proportions in harvestable seed and in all seed, which includes small seed that would be lost in combining.

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Figure 3. Proportion of midge-damaged seed in CGC samples, 2010: evaluation by CGC personnel and actual damage assessed by Ian Wise.

Figure 4. 1000 kernel weights (g) of undamaged and damaged harvestable seed from dissected spikes, 2010.

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Figure 5. 1000 kernel weights (g) of undamaged and damaged seed from cleaned CGC samples, 2010.

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Summary:

Midge damage was considerably less in 2009 (year 3) and in 2008 (year 2), compared to 2007 (year 1) and 2010 (year 4) (see Appendix B, Fig. 6).

Results from years 1, 2, 3 and 4 showed that the resistant blends varied in their effectiveness

against midge, but generally midge damage to kernels is less in cultivars with the Sm1 gene (varietal blends), BW394 VB showing the least proportion of seeds damaged by midge feeding across all locations and years. Market grade is expected to be generally higher for those varieties possessing Sm1 than for varieties that lack this gene. Waskada, one of the susceptible varieties, consistently showed lower damage than the other susceptible cultivars across locations and years.

Cultivars carrying the Sm1 gene should not be considered as midge tolerant. In host-plant

resistance terminology, tolerance means that plants can tolerate or recover from feeding injury without any significant loss of yield and without any effect on the insect pest. The midge resistant lines in study are the complete opposite, as they kill the pest (antibiosis) but have varying degrees of intolerance to feeding injury. The three cultivars BW362, BW365 and BW841 obviously have not achieved the gold standard of resistance (pest control without crop injury), but lines like BW394 clearly indicate it is possible. We also should label the ‘susceptible’ cultivar Waskada as being partially resistant, but in this case it is antixenotic resistance. This type of resistance is based on the plants being less preferred than susceptible types. These are the correct biological terms, which, admittedly, may or may not translate well to the agriculture community. For communication to the larger agricultural community, the term tolerance is being used, with the indication that a reduced level of midge damage is expected (the term resistance is often being equated with immunity, which is not the case with midge resistance at the present levels).

A new (other than SNP) marker has been identified, which is being used to monitor the

proportion of resistant and susceptible components for each of the four varietal blends being studied (Appendix C). This new marker can be used to fingerprint all 4 blends (ie. use only a single marker for all 4 blends scoring). After three years of analysis, the blend ratios showed the expected 90% resistant: 10 % susceptible, within sampling error. Some locations/genotypes showed a 95:5 or 85:15 ratio which could be due to a very small genetic (cultivar) drift, but most probably to sampling error. These apparent departures from the expected 90:10 ratio will be monitored more closely in future generations.

End-use suitability analysis for year 1 and 2 has been completed, and for year 3 is

progressing (Appendix D). Overall, an increase in midge damage percent significantly decreased kernel weight, kernel length and diameter, and falling number value in both the resistant wheat blends and the susceptible wheat cultivars. Midge damage (up to 20%) did not affect kernel hardness, kernel and flour redness and dough strength of the resistant blends. But midge damage caused harder kernels, less red kernel and flour colour, and weaker dough strength in susceptible cultivars.

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APPENDIX A AGRONOMIC DATA

Fig. 1. Plant height of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010.

40

60

80

100

120

140

BRA MEL INH REG SAS SWC LET LAC

Pla

nt h

eigh

t (cm

)

BW362 VBBW365 VBBW394 VBBW841 VBKatepwaCDC TealWaskadaAC Intrepid

18

Fig. 2. Heading of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 4 locations in the prairie provinces of Canada in 2009.

40

45

50

55

60

65


Hae

din

g (

day

s fr

om

see

din

g)


19

Fig. 3. Maturity of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010.

80

85

90

95

100

105

110

115

120

125

130

135


Mat

uri

ty (

day

s fr

om

see

din

g)


20

Fig. 4. Lodging of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010.

0

1

2

3

4

5

6

7

8

9


Lo

dg

ing

(1-

9)

BW362 VBBW365 VB

BW394 VBBW841 VBKatepwaCDC Teal

WaskadaAC Intrepid

21

Fig. 5. Seed yield of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010.

0

2000

4000

6000

8000

10000


See

d y

ield

(kg

ha

-1)

BW362 VBBW365 VBBW394 VBBW841 VB

KatepwaCDC TealWaskadaAC Intrepid

22

Fig. 6. Percent dockage of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2009.

0

1

2

3

4

5

6

7


Do

ckag

e (%

)

BW362 VB

BW365 VB

BW394 VB

BW841 VB

Katepw a

CDC Teal

Waskada

AC Intrepid

23

Fig. 6. Seed weight of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2009.

20

25

30

35

40

45


Seed weight (g 1000-1)

BW362 VB

BW365 VB

BW394 VB

BW841 VB

Katepwa

CDC Teal

Waskada

AC Intrepid

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APPENDIX B

MIDGE DAMAGE ASSESSMENT FROM WHEAT SPIKES

Fig. 1. Seeds per spike of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

20

25

30

35

40

45


See

ds

spik

e-1

BW362VBBW365VBBW394VBBW841VBKatepw aCDC TealWaskadaAC Intrepid

25

Fig. 2. Seed weight (undamaged seeds) of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

10

15

20

25

30

35

40

45


Un

dam

aged

see

d w

eig

ht

(g 1

000

seed

s-1

)


26

Fig. 3. Percentage of harvestable damaged seeds of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

5

10

15

20

25

30

35


Har

vest

able

dam

aged

see

d (%

)


27

Fig. 4. Percentage of not harvestable damaged seeds (seeds potentially lost) of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

5

10

15

20

25

30

35


No

n h

arve

stab

le d

amag

ed s

eed

s (%

)


28

Fig. 5. Percentage of total damaged seed of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

5

10

15

20

25

30

35


Tota

l dam

aged

see

d (%

)


29

Fig. 6. Percentage damaged seed of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2007, 2008, 2009 and 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

10

20

30

40

50

60

BRA MEL INH REG SAS SWC LET LAC BRA MEL INH REG SAS SWC LET LAC BRA MEL INH REG SAS SWC LET LAC BRA MEL INH REG SAS SWC LET LAC

Total damaged seed (%)

BW362 VB

BW365 VB

BW394 VB

BW841 VB

Katepwa

CDC Teal

Wascada

AC Intrepid

2007 2008 2009 2010

30

Fig. 7. Percentage of estimated yield lost of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

5

10

15

20

25


Est

imat

ed y

ield

loss

(%)


31

Fig. 8. Percentage of estimated yield lost of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2007, 2008, 2009 and 2010. Data generated from 20 random spikes collected from each plot at all locations.

0

5

10

15

20

25

30

35

40

45

BRA MEL INH REG SRC SWC LET LAC BRA MEL INH REG SRC SWC LET LAC BRA MEL INH REG SAS SWC LET LAC BRA MEL INH REG SAS SWC LET LAC

Estimated yield loss (%)

BW362VB

BW365VB

BW394VB

BW841VB

Katepwa

CDC Teal

Waskada

AC Intrepid

2007 2008 2009 2010

32

MIDGE DAMAGE ASSESSMENT FROM HARVESTED GRAIN SAMPLES

Fig. 9. Percent midge damage of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) grown at 8 locations in the prairie provinces of Canada in 2010. Assessment done at the Canadian Grain Commission (CGC) from cleaned harvested seed samples from each plot and location.

CGC assessment

0

2

4

6

8

10

12


Damaged seed by CGC (%)

BW362VB

BW365VB

BW394VB

BW841VB

Katepwa

CDC Teal

Waskada

AC Intrepid

33

Fig. 10. Percent midge damage of 4 midge resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties (Katepwa, CDC Teal, Waskada and AC Intrepid) at 8 locations in the prairie provinces of Canada in 2010. Assessment done at the Cereal Research Centre (CRC) from cleaned harvested seed samples from each plot and location.

CRC assessment

0

2

4

6

8

10

12


Damaded seed by Ian (%)

BW362VB

BW365VB

BW394VB

BW841VB

Katepwa

CDC Teal

Waskada

AC Intrepid

34

APPENDIX C

Development of SNP markers and DNA analysis to assess drift of SM1 gene (Funded by WGRF)

The "marker discovery" phase for the 4 varietal blends has been successfully completed. These 4 varietal blends were initially described in the original ADF grant and the first generation refuge seed from 8 locations is currently being tested (~13,000 tests).

The Invader scoring platform for all varietal blends is very robust and reliable (see attachment examples, varietal blend #4; varietal blend #3( Melfort); varietal blends 1&2). We use 96-well plates and single seeds for the accurate quantitation ( 90:10% ratio) of the midge varietal blends ( 384, 1256-well plates can also be used, or even the 186,000- well tape array).

First generation varietal blends: Each plot (100 seeds) hovers around the expected 90%R : 10 %S with the average for the particular location ( 4- plots of about 400 seeds tested) being even closer to the 90%R :10% S ratio. This is true for all the varietal blends and locations tested so far (see attachment - midge locations). Conclusion: There was very little , if any drift in the constitution of the varietal blends after a single generation: evolution does not occur overnight. Farmers can save their first generation seed. Second generation varietal blends: The stability of the blends was monitored over two generations. A total of ~ 29,000 individual seeds were scored for both the 1st & 2nd generations (100 seeds per plot/ 4-5 plots per location/ 8 locations/ 4 genotypic blends). Conclusion: The blend ratios showed the expected 90% resistant: 10 % susceptible, within sampling error (See graphs in following pages). Some locations/ genotypes showed a 95:5 or 85:15 ratio which could be due to a very small genetic (cultivar) drift or sampling error………. probably sampling error. We have developed a new & improved marker that can fingerprint all 4 blends, ie. use only a single marker for all 4 blends scoring. This new marker and a different marker-scoring platform showed a 98-99% concordance (identical results) with the former Invader SNP scoring system. Thus we believe all the ratio data is correct and reliable.

35

Third generation varietal blends: All completed! DNA fingerprinting of a total of ~46,000 individual seeds scored (100 seeds per plot/4-5 plots per location/ 8 locations/ 4 genotypic blends/ 3 generations) was done. Only 3 out of 96 samples (8 locations X 4 blends X 3 generations) were outside of the expected 90% resistant:10% susceptible range (see attachment values, Midge DSS Result, RED arrows). The acceptable ranges are the 85%: 15% or the 95%: 5% ranges. This is a 3.1% level, well within the acceptable 95% confidence interval-- +/- 2 standard deviations from the mean will lead to 5% (1 in 20) of samples being assessed as inferior by chance alone. From these observations, CRC’s SNP scoring of blends is reliable & accurate and CSGA can agree to certify varietal blends based on the 95% confidence interval. There was no obvious drift, upwards or downwards change from the 90:10 % ratio, over 3 generations within the sampling error. Breeders seed purity (contamination of original breeders seed):

We tested the % contamination of the original breeders seed using the SNP-DNA markers for each line in the varietal blend.

Line # seeds tested % contamination

Unity (BW362) 576 1 %

Waskada (BW357) 288 1.6%

Fieldstar (BW365) 480 0 %

Goodeve (BW841) 192 1.6 %

Intrepid 192 0 %

Whether using single seeds from a “bag of seeds” or individual plots that contribute to the breeders seed, the breeders seed purity ranged from 98.4- 100% on our sample size. This is not a “foolproof” test since contamination by another cultivar that has the same identical SNP-allele at the SNP site as the line tested cannot be distinguished.

36

Fourth generation varietal blends: This work is still under progress. Additional funding is necessary to complete this work.

SNP-DNA markers for a new varietal blend- Vesper VB:

Markers for Vesper VB that has the 90% resistant line (BW415- SNP A allele) and 10% susceptible line ( Waskada- G allele) have been discovered. This blend will be commercialized by SeCan seed company.

High-speed SNP-DNA fingerprinting in seeds using the Invader-Plus genotyping assay:

This assay is used for high-throughput & high-speed genotyping (see attachment). The assay is performed entirely in a microtiter format with no electrophoresis or DNA purification steps. The reporter system can be dried down to the surface of the microtiter plate wells with only the addition of the PCR template required. There is no growing of plants/seedlings. The assay uses a single, closed microwell where a continuous reaction for both the PCR & Invader components is employed. The total assay time from “seed to base call” is under two hours, from 1000 to 10,000 assays can be completed in a single day and approximate costs are $0.30 to $0,70 per sample per SNP site.

37

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

2007- 09 MIDGE BW841(GOODEVE) VB RESULTS

07 BW841 07 SUS 08 BW841 08 SUD BW841 BW SUS

07 BW841 90.91 86.75 89.49 93.08 88.98 90.14 89.13 86.90

07 SUS 9.09 13.25 10.51 6.92 11.02 9.86 10.87 13.10

08 BW841 86.98 90.42 89.77 91.77 93.28 93.01 90.87 93.03

08 SUD 13.02 9.58 10.23 8.23 6.72 6.99 9.13 6.97

BW841 89.08 90.49 89.74 91.01 89.72 92.44 89.73 88.31

BW SUS 10.92 9.51 10.26 8.99 10.28 7.56 10.27 11.69

BrandonIndian Head

Melfort SaskatoonSwift

CurrentLethbridge Lacombe Regina

38

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

2007- 09 MIDGE BW394VB (SHAW) RESULTS

07 SUS 07 BW394 08SUS 08BW394 09SUS 09BW394

07 SUS 10.84 11.05 9.73 8.22 7.38 6.82 7.98 10.66

07 BW394 89.16 88.95 90.27 91.78 92.62 93.18 92.02 89.34

08SUS 7.60 7.60 11.16 14.65 8.19 10.61 10.21 7.77

08BW394 92.40 92.40 88.84 85.35 91.81 89.39 89.79 92.23

09SUS 11.30 9.87 9.11 7.68 5.02 14.05 7.56 8.26

09BW394 88.70 90.13 90.89 92.32 94.98 85.95 92.44 91.74

Brandon Indian Head Melfort Saskatoon Swift Current Lethbridge Lacombe Regina

39

Bra

ndon

Bra

ndon

Bra

ndon In

dian

Hea

d

Indi

an H

ead

Indi

an H

ead

Mel

fort

Mel

fort

Mel

fort

Sas

kato

on

Sas

kato

on

Sas

kato

on

Sw

ift C

urre

nt

Sw

ift C

urre

nt

Sw

ift C

urre

nt

Leth

brid

ge

Leth

brid

ge

Leth

brid

ge

Laco

mbe

Laco

mbe

Laco

mbe

Reg

ina

Reg

ina

Reg

ina

0

10

20

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40

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60

70

80

90

100

2007- 2009 MIDGE DSS TRIAL BW365 VB ( FIELDSTAR ) RESULTS

07 DSS BW365VB 07 SUS 08 DSS BW362VB 08 SUS 09 DSS BW365VB 09 SUS

07 DSS BW365VB 82.2147651 97.40634006 91.66666667 92.46987952 88.39779006 91.86046512 88.81789137 89.3982808

07 SUS 17.7852349 2.593659942 8.333333333 10.24096386 11.60220994 8.139534884 11.18210863 10.6017192

08 DSS BW362VB 94.67849224 95.49356223 94.98956159 91.84549356 84.95575221 90.51918736 91.81034483 87.47252747

08 SUS 5.321507761 4.506437768 5.010438413 8.154506438 15.04424779 9.480812641 8.189655172 12.52747253

09 DSS BW365VB 94.22222222 91.64835165 94.19642857 89.97867804 85.03253796 93.79157428 95.404814 92.69406393

09 SUS 5.777777778 8.351648352 5.803571429 10.02132196 14.96746204 6.208425721 4.595185996 7.305936073


40

Bra

ndon

Bra

ndon

Bra

ndon

Indi

an H

ead

Indi

an H

ead

Indi

an H

ead

Mel

fort

Mel

fort

Mel

fort

Sas

kato

on

Sa

skat

oon

Sas

kato

on

Sw

ift C

urre

nt

Sw

ift C

urr

ent

Sw

ift C

urre

nt

Leth

brid

ge

Let

hbr

idge

Leth

brid

ge

Laco

mbe

Lac

omb

e

Laco

mbe

Reg

ina

Reg

ina

Reg

ina

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2007- 2009 MIDGE DSS TRIAL BW362 VB (UNITY) RESULTS

07 DSS BW362VB 07 sus 08 DSS BW362VB 08 SUS 09 DSS BW362VB 09 SUS

07 DSS BW362VB 94.36619718 94.00630915 93.4537246 90.40178571 90.9512761 92.49448124 91.23595506 92.95154185

07 sus 5.633802817 5.993690852 6.546275395 9.598214286 9.048723898 7.505518764 8.764044944 7.04845815

08 DSS BW362VB 91.83098592 95.07692308 91.6967509 96.40883978 90.13333333 94.00544959 93.93939394 91.68975069

08 SUS 8.169014085 4.923076923 8.303249097 3.591160221 9.866666667 7.084468665 8.484848485 8.310249307

09 DSS BW362VB 92.04819277 94.58823529 95.215311 92.80898876 89.89010989 92.03747073 93.0875576 91.91011236

09 SUS 7.951807229 5.411764706 4.784688995 7.191011236 10.10989011 7.962529274 6.912442396 8.08988764


41

APPENDIX D End-use suitability (quality) Analysis

(Funded by MII)

Midge Quality Project Odean Lukow and Dora Fenn, (13 January 2011) End-use quality characteristics were studied from three wheat midge resistant varietal blends (97B64-F9A3+Waskada (BW362), 97B64M1B3+Waskada (BW365) and BW841+AC Intrepid, in a 9 to 1 ratio), containing the midge resistance antibiosis gene Sm1, and 3 midge susceptible cultivars (CDC Teal, Waskada, AC Intrepid), lacking the Sm1 gene. All samples were grown in 2007, 2008 and 2009 at 8 locations in Manitoba, Saskatchewan and Alberta. Four field replicates were combined into 2 (rep 1+2 and rep 3+4) in this study. A portion of seeds in each sample was kept as ‘as-is’. The remainder was hand separated into midge damaged and undamaged seeds, followed by reconstituting samples to various levels of midge damage (by weight). A total of 180 seed samples representing 6 cultivars, 2 replicates, 3 locations (Lacombe, Saskatoon, Melfort) and 5 midge damage groups (‘as-is’, 0, 2, 10 and 20%) were studied for kernel characteristics, flour milling quality, dough properties, falling number and total soluble phenolics content. Results from 2007 Results from the midge resistant and susceptible wheat types were analyzed separately in a mixed model using SAS with replicate and location as random variables. No significant differences were observed among the five midge damage groups in both wheat types for seed and straight-grade flour brightness and yellowness or for flour milling yield percent (data not shown). Attached figures show the averages of end-use quality, combined over location and blends/cultivars, for each damage group and wheat type. Significant differences among the five midge damage groups were observed in the resistant and susceptible wheat types for kernel weight, kernel length and diameter (Single kernel characterization system), falling number value, and in the susceptible wheat type for kernel hardness, seed and straight-grade flour redness, mixograph energy after peak, total energy and total band width energy, and in the resistant wheat type for total soluble phenolics content. Overall, an increase in midge damage percent significantly decreased kernel weight, kernel length and diameter, and falling number value in both the resistant wheat blends and the susceptible wheat cultivars. An increase in percent midge damage in the susceptible wheat cultivars significantly increased kernel hardness and decreased kernel and flour redness and dough strength, as evidenced by mixograph energy after peak, total energy, and total band width energy. Up to 20% midge damage in resistant blends showed no significant difference in kernel hardness, kernel and flour redness and mixograph dough strength compared to the undamaged seeds. For thousand kernel weight, falling number, seed redness, and mixograph energy after peak, significant differences between ‘as-is’ and 0% damage groups were shown for the susceptible cultivars and not for the resistant blends. Significantly higher amount of total soluble phenolics was detected only from resistant blends with 20% midge damage. Further HPLC assay for individual phenolic will be performed on all resistant blends and susceptible cultivars.

42

1000 kernel weight

a

a

a

ba

ab

ac

b

d

2527293133353739

Resistant SusceptibleWheat Type

(g)

Clean

As-is

2% Damage

10% Damage

20% Damage

Single Kernel Characterization System

aa

aba

aba bc

b dc

20

25

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45


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nel

Wt

(mg

)

Clean

As-is

2% Damage

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20% Damage


a

aa

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a

b

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b

c

400

410

420

430

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450


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nel

Len

gth

(m

m)

Clean

As-is

2% Damage

10% Damage

20% Damage


aa

aab

a

a

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2.4

2.6

2.8

3.0


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nel

Dia

met

er

(mm

)

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As-is

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10% Damage

20% Damage

Falling Number

a

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c

250

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Sec

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a a a

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545658606264666870


Ker

nel

Har

dn

ess

Clean

As-is

2% Damage

10% Damage

20% Damage

Seed Redness

a

bc

NS

abbc

c

3.10

3.15

3.20

3.25

3.30

Resistant Susceptible

Wheat Type

a* V

alu

e

Clean

As-is

2% Damage

10% Damage

20% Damage

Flour Redness

a a

NSa

ab

b

-0.80

-0.76

-0.72

-0.68

-0.64

-0.60


a* V

alu

e

Clean

As-is

2% Damage

10% Damage

20% Damage

43

Mixograph

a

bbc

NS

cbc

420440

460480500

520

540560580


En

erg

y af

ter

pea

k (%

*min

)

Clean

As-is

2% Damage

10% Damage

20% Damage

Mixograph

a

ab

bc

NS

cbc

550

570

590

610

630

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670

Resistant Susceptible

Wheat Type

To

tal E

ner

gy

(%*m

in)

Clean

As-is

2% Damage

10% Damage

20% Damage

Mixograph

aababc

NS

bc c

200

220

240

260

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300


To

tal b

and

wid

th

ener

gy

(%*m

in) Clean

As-is

2% Damage

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20% Damage

Total Soluble Phenolics Content

a aa

NS

a

b

1080

1130

1180

1230

1280


ug

/g f

lou

r

Clean

As-is

2% Damage

10% Damage

20% Damage

Bars indicate averages of end-use quality; NS = non-significant; bars with different letters represent significant differences (P≤ 0.05).

Results from 2008 The 2008 midge samples from all locations were collected. Indian Head, Regina, and Saskatoon samples, which had low, medium and high midge damage percent respectively, were chosen for the 2008 quality study. Samples were sorted by hand into midge damaged and undamaged groups. Due to less midge occurring in 2008, only 3 treatment groups could be formed: ‘as-is’, clean and 2% midge damage. Quality analyses were completed for the 2008 samples and included: kernel weight, test weight, seed colour, kernel hardness, wholemeal colour, straight-grade colour, Brabender flour yield, falling number value, total phenolics, phenolics content on HPLC, alpha-amylase, ash content, mixograph parameters and extension properties. Statistical analyses will be run to determine quality differences between wheats with varying midge damage. Results from 2009 The 2009 midge samples from all 8 locations were collected. Three locations: Indian Head, Melfort and Brandon, were chosen for the 2009 quality study. Samples (144 total) are currently being sorted by hand into the following groups: ‘as-is’, clean, 2% midge damage and 10% midge damage. Quality analyses will be done as time and funding permits. Results from 2010

spopp

Typewritten Text

spopp

Typewritten Text

spopp

Typewritten Text

The seed samples collected from all 8 locations of the midge study in 2010 were not requested for quality analyses.

spopp

Typewritten Text

spopp

Typewritten Text

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Typewritten Text

1

Relative performance of four midge-resistant wheat varietal blends in Western Canada

C. L. Vera1, S. L. Fox2, R. M. DePauw3, M. A. H. Smith2, I. L. Wise2, F. R. Clarke3, J. D. Procunier2 and O. M. Lukow2

1Agriculture and Agri-Food Canada, Melfort Research Farm, P.O. Box 1240, Melfort, SK, Canada S0E 1A0 (e-mail: [email protected]); 2Agriculture and Agri-Food Canada, Cereal Research Centre, 195 Dafoe Road, Winnipeg, MB, Canada R3T 2M9; 3Agriculture and Agri-Food Canada, Semiarid Prairie Agricultural Research Centre, P.O. Box 1030, Swift Current, SK, Canada S9H 3X2.

Final report to ADF

This document will also be submitted to the Canadian Journal of Plant Science for publication.

SUMMARY

Orange wheat blossom midge (OWBM), Sitodiplosis mosellana (Géhin), causes significant yield losses to spring wheat in western Canada in severe infestations. To mitigate losses, midge-resistant wheat varietal blends, consisting of cultivars with the Sm1 midge resistance gene and 10% interspersed midge susceptible refuge, have been made available to farmers. To test their performance relative to conventional midge-susceptible cultivars, four varietal blends (Fieldstar VB, Goodeve VB, Shaw VB and Unity VB) were grown during four growing seasons (2007-2010), at eight locations in the provinces of Manitoba (Brandon), Saskatchewan (Indian Head, Melfort, Regina, Saskatoon and Swift Current) and Alberta (Lacombe and Lethbridge), and compared to four conventional, midge-susceptible cultivars (AC Intrepid, CDC Teal, Katepwa and Waskada). Midge damage was higher in 2007 and 2010 than in 2008 and 2009. In general, the varietal blends, as a group, yielded more grain than the susceptible cultivars, especially when grown in environments with high OWBM pressure (12.8% seed damage). In environments with low OWBM pressure (0.9% seed damage), the varietal blend yield increases were smaller but still significant, indicating that some of the varietal blends had additional superior attributes, besides OWBM resistance.

2

INTRODUCTION Orange wheat blossom midge (OWBM), Sitodiplosis mosellana (Géhin), was first detected in western Canada (Manitoba) in 1901 by Norman Criddle (Fletcher 1902) and was well established in that province by 1954 (Barker 1984) and in Saskatchewan by 1983 (Olfert et al. 1985). Outbreaks of this pest in North America on wheat and other cereal crop species (Wright and Doane 1987) have occurred (Olfert et al. 1985), since it was first identified in Quebec in 1828 (Felt 1921). Feeding by larvae on a developing wheat kernel causes it to shrivel and become deformed. Loss of shriveled kernels reduces yield, whereas retained damaged kernels can reduce grade and end-use quality performance, as well as germination and early growth rate of newly germinated seedlings (Lamb et al. 2000). Genetic antibiotic resistance to OWBM was first detected in winter wheat by Barker and McKenzie (1996). Resistance was identified as a single antibiotic gene named Sm1, on chromosome 2BS (Thomas et al. 2005), in a study to transfer this gene into spring wheat (McKenzie et al. 2002). This gene is being used in western Canada to create midge-resistant spring wheat cultivars, by selecting directly for response to OWBM or indirectly using molecular markers linked to the gene, followed by bioassay to confirm resistance (DePauw et al. 2009; Fox et al. 2010). The first three wheat lines carrying the Sm1 gene were supported for registration in the 2007 meeting of the Prairie Recommending Committee for Wheat, Rye and Triticale (PRCWRT).

Resistance to insect pests based on a single gene is often short-lived, because of the selection of virulent insect biotypes. This has been observed in Hessian fly, a close relative of the OWBM (Gallun 1977, Foster et al. 1991). To preserve host-plant resistance, susceptible plants are grown among (interspersed refuge) or in proximity to (separate refuge) resistant plants. This was developed and is now required in commercially grown cotton and corn in U.S. to protect the efficacy of Bacillus thuringiensis (Bt) technology (Carrière et al. 2005). The use of an interspersed refuge is now being incorporated into wheat in the form of varietal blends, to protect the effectiveness of the Sm1 gene (Smith et al. 2004, 2007).

Varietal blends in a ratio of 90% midge-resistant wheat and 10% susceptible wheat are now commercially sold in the Canadian seed market. An interspersed refuge to manage virulence in OWBM populations was adopted because adult OWBM tend not to move far from their sites of emergence prior to mating. A 10% interspersed refuge produces enough avirulent adults to ensure that nearly all virulent OWBM would likely mate with an avirulent individual, thus discouraging the selection of virulence in the OWBM population, especially if the inheritance of the resistance is recessive, where the hybrid offspring would not survive when feeding on resistant plants (Tabashnik et al. 2008).

This study was designed to evaluate, in different environments (year-locations) with variable weather conditions and dissimilar midge pressures, the effectiveness of the Sm1 gene in protecting wheat against loss of market grade and grain yield by the OWBM.

MATERIALS AND METHODS Treatments

3

Varietal blends of four midge-resistant cultivars carrying the Sm1 gene, each containing a 10% refuge, and four midge-susceptible check cultivars were grown for four consecutive years (2007- 2010) at eight locations in the three prairie provinces of western Canada (Table 1). Treatments were as follows: Goodeve VB [blend of Goodeve (DePauw et al. 2009) (90%) and AC Intrepid (10%)] Fieldstar VB [blend of Fieldstar (Fox et al. 2012) (90%) and Waskada (10%)] Shaw VB [blend of Shaw (CFIA 2011) (90%) and BA51*C222 (10%)] Unity VB [blend of Unity (Fox et al. 2010) (90%) and Waskada (10%)] AC Intrepid (DePauw et al. 1999) CDC Teal (Hughes and Hucl 1993) Katepwa (Campbell and Czarnecki 1987) Waskada (Fox et al. 2009) The four varietal blends were reconstituted annually, using a common seed source for all locations each year. A randomized complete block design with four replications was used at each location. The following agronomic parameters were measured or visually estimated: days to heading, days to maturity, plant height, lodging, seed yield, seed weight and dockage. Seed yield constituted cleaned seed that did not pass through a sieve with triangular (inscribed circle with a diameter of 2.26 mm) perforations, using a Carter-Day dockage tester. Chaff, seed and other impurities which passed through this sieve were considered dockage. Locations and other information

The experiment was seeded at one location in Manitoba (Brandon), five locations in Saskatchewan (Indian Head, Melfort, Regina, Saskatoon and Swift Current) and two locations in Alberta (Lacombe and Lethbridge), usually in May. Seeding dates and other location and plot information are listed in Table 1. The experiment was started in the 2007 growing season because high levels of OWBM infestation were forecasted for that year.

Plot equipment used for seeding and harvesting these trials was similar at all locations. A target of 220 viable seeds m-2 was planted in plots of sizes listed in Table 1. Adequate fertilization regimes, based on soil tests, were applied at each location, targeting a total (soil test plus applied) of a minimum of 80-100 kg ha-1 of nitrogen (with lower rates in drier locations) and 60 kg ha-1 of phosphorus (Table 1). Supplemental irrigation was applied to the trials grown at Lethbridge in the amounts of 178, 127, 102 and 51 mm, during the four years of experimentation, respectively. Ten spikes were collected from each treatment, replication and environment (year-location) at the hard dough stage to assess the level of OWBM damage (OWBM pressure) on the developing seeds. These spikes were dissected, the number of midge-damaged and undamaged seeds counted, and the percentage of midge-damaged seeds calculated for each treatment in each environment. These percentages were used to categorize environments according to different levels of infestation (Table 2). Full data and analysis of the spike samples will be presented elsewhere.

4

Growing season weather information for the different environments is found in Table 3. Data analysis Environments (year-locations) were assigned to one of three groups based on levels of midge damage measured from collected spikes (Table 2). To establish the three damage level groups and minimize Standard Deviations (S.D.s), all 32 year-locations were sorted from highest to lowest mean percentage of midge-damaged seed. The five highest and five lowest percentages were classified as “high midge pressure” and “low midge pressure” groups, respectively, with the remaining classified as “moderate midge pressure”. S.D.s were calculated and compared for each group. Then the highest and lowest percentages from the “moderate” group were re-classified to the “high” and “low” groups, respectively and S.D.s recalculated. As this process was repeated, the S.D.s for the “high” and “moderate” groups were observed to decline while the S.D. of the “low” group increased. At the final accepted classification, the S.D.s of the low and medium groups were equal.

SAS [version 8.2, Littell et al. (1996)] was used to perform all analyses. To check for the influence of individual observations and possible outliers, studentized residuals were examined (Rawlings, 1988, p.250). The PROC MIXED procedure was used for the mixed model analysis of variance of the agronomic parameters, within each of the three midge-pressure groups. To describe the response of each treatment in the environments, cultivars were considered fixed, while replications within environments, environment and its interaction with cultivar were considered random. For each agronomic parameter, comparisons among least-squares means of treatments were made, and the appropriate standard errors of differences between means were calculated using the DIFF option. ESTIMATE statements were used to compare the groups of treatments with and without the Sm1 allele.

RESULTS AND DISCUSSION The combined analysis over all 32 environments (year-locations), categorized into three levels of OWBM damage, indicated that environment was significant for almost all, and cultivar and the interaction of environment with cultivar were significant for all agronomic traits measured (Table 4). However, as the performance of the cultivars relative to each other was similar when averaged in each environment, their overall averages were combined over each of the three groups of environments. Heading and Maturity Heading and maturity were measured at 20 and 27 of 32 environments, respectively, with the missing data distributed across the three midge-pressure groups. It was observed that heading and especially maturity were strongly influenced by air temperature. For example, the number of days to heading and to maturity were both lowest in 2007, the warmest of all four years of experimentation, with 1551 growing degree days (GDD), compared to the four-year average of 1447 GDD (Table 3). Heading was also influenced by seeding date. For example, plots at Saskatoon took the least time to start heading (49

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days from seeding), which was probably the result of delayed seeding at this location each year (Table 1). Maturity may also have been affected by soil moisture. Wheat plants took longest to mature (118 days from seeding) at the Lethbridge site which normally received supplementary irrigation.

Although cultivars did not show the same heading and maturity pattern in all environments, AC Intrepid was consistently the earliest cultivar to head and to mature in most environments where these parameters were measured. Goodeve VB, Katepwa and CDC Teal showed medium maturity, while Fieldstar VB, Shaw VB, Unity VB and Waskada were generally the latest in maturity (Table 5).

Heading and maturity were earlier for the environments with high OWBM pressure (Table 5). This was not a direct effect of OWBM pressure on these two traits but rather similar environmental characteristics that favour both earlier maturity and successful egg laying by the midge. Most locations in 2007 were under high OWBM pressure. They were characterized by being seeded later than in other years and the growing season tended to be slightly warmer and drier. Height Plant height was measured in all environments. Both air temperature and soil moisture (precipitation) seem to have affected this parameter as well. For example, the shortest plant height values were recorded at Swift Current (83.1 cm) and Regina (86.8 cm), locations with normally low precipitation and relatively high GDD values (Table 3).

Averaged across environments, Fieldstar VB, Katepwa, Shaw VB, Unity VB and Waskada were tallest, while AC Intrepid, CDC Teal and particularly Goodeve VB were shortest. This pattern was observed in all three midge-pressure groups (Table 5). Plant height is a highly heritable trait, showing a similar relative trend from site to site and from year to year (Iqbal et al. 2006).

Lodging Lodging was an infrequent occurrence in most environments during this study (data not shown). However, Lethbridge reported severe lodging in 2007 and 2009 and not as severe (low to moderate) in 2008 and 2010. These lodging events were probably favoured by the supplementary moisture supplied by irrigation at this site each year, as well as by high soil N availability, especially in 2007. Low to moderate lodging was also observed at Brandon in 2009 and 2010, Melfort in 2007 and 2009, Regina in all four years, and Saskatoon in 2008 and 2009. Goodeve VB and Shaw VB showed lower lodging values in these environments, when compared to other treatments, especially when lodging was most severe. Yield Grain yield was collected from all environments. Highest mean yield values were obtained in 2009 (4814 kg ha-1) and lowest in 2007 (3193 kg ha-1). The year 2007 was the warmest (104% of the normal) and also the second driest (96% of normal) of the four years of experimentation (Table 3). This negatively affected seed yield, especially in locations like Swift Current, which in 2007 received only 52% of normal precipitation, resulting in an average seed yield of only 1299 kg ha-1, compared to the total average of 3193 kg ha-1, obtained for all combined locations that year (data not shown).

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Averaged over all years and cultivars, Lacombe had the highest average seed yield (6201 kg ha-1) and Swift Current the lowest (2587 kg ha-1). Lacombe had much higher (141%) than normal precipitation in the dry 2007 and it was also warmer (107% of normal GDD) that year (Table 3).

In general, the midge-resistant varietal blends, as a group, yielded significantly (P < 0.01) higher than the midge-susceptible cultivars in all levels of OWBM infestation (Table 6), with Shaw VB and Unity VB yielding significantly (P < 0.05) higher than the other varietal blends(Table 5). The significant yield advantage of the varietal blends in the low midge damage environments provides evidence that the presence of Sm1 does not negatively impact yield. Within the susceptible cultivars, Waskada, and occasionally AC Intrepid, yielded significantly (P < 0.05) higher than the other midge-susceptible cultivars, and in environments with low OWBM pressure Waskada yielded as high (P < 0.05) as Shaw VB and Unity VB. On the other hand, the midge-resistant varietal blend Goodeve VB did not show a yield advantage over some of the midge-susceptible cultivars, including its male parent AC Intrepid, but especially Waskada, even in environments with high OWBM pressure (Table 5). This is likely due to Goodeve VB maturing significantly earlier than Waskada and to Waskada exhibiting oviposition deterrence (antixenotic resistance) to wheat midge which reduces midge damage through reduced larvae numbers (Fox et al. 2009). Time to maturity is known to be positively related to grain yield (Iqbal et al. 2007). The older cultivar Katepwa yielded significantly (P < 0.05) lower than any other cultivar used in this study, in all levels of OWBM infestation.

Waskada, the 10% interspersed refuge in both Fieldstar VB and Unity VB, on average yielded significantly less than these two varietal blends under high midge pressure (Table 5). Under moderate midge pressure, Waskada yielded significantly less than only Unity VB, and under low midge pressure its yield was not significantly different from either varietal blend. However, AC Intrepid, the 10% interspersed refuge in Goodeve VB, on average yielded similar to Goodeve VB at all levels of midge pressure.

A comparison of the mean yield advantage of cultivars having Sm1 over the mean yield of the susceptible cultivars in each of the midge damage level groups (Table 6) provides some indication as to the value of this resistance gene. In the low and medium damage groups, there is a 4.0% and 5.5% yield advantage, respectively, which provides an indicator of yield improvement unrelated to the occurrence of losses caused by OWBM. However, in the high damage group, there is a 14.8% yield advantage (473 kg/ha advantage of Sm1 cultivars / 3188 kg ha-1 of susceptible cultivars). Thus it may be conjectured, that about 11% of this advantage is due to the presence of Sm1 and 4% of the advantage is due to other gains made through plant breeding. Seed weight and Dockage Seed weight was measured in all environments and, being a seed yield component, it was affected by factors such as precipitation and temperature, OWBM pressure, and inherent cultivar differences. Seed weight was highest in 2009 (37.5 mg seed-1) and lowest in 2007 (31.4 mg seed-1). It was also highest at Lacombe (39.0 mg seed-1) and lowest at Swift Current (30.5 mg seed-1). Of the four varietal blends and four susceptible cultivars, AC Intrepid had the heaviest seeds (38.0 mg seed-1) and Fieldstar VB the lightest (31.6 mg

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seed-1). This pattern was observed in all three groups of environments (Table 5). Seed weight was also lower (33.0 mg seed-1) in environments with high OWBM pressure (12.8% seed damage) than in environments with lower OWBM pressure (34.2 and 36.6 mg seed-1 in environments with 3.8% and 0.9% seed damage, respectively).

Dockage was negatively and significantly (P < 0.001) correlated to seed size (correlation coefficients of -0.81 **, -0.92 *** and -0.79 * for the three groups of environments, respectively), as seed was the main component of dockage. This was observed in Swift Current in 2007, where seeds were very small (19.7 mg seed-1), which resulted in very high dockage (15%) compared to the other locations that year. As a group, the varietal blends had significantly greater dockage than the susceptible cultivars (Table 6), largely due to the high dockage in Fieldstar VB and Unity VB (Table 5). Among the cultivars, the resistant varietal blend Fieldstar VB had the smallest seeds and also showed the highest dockage (3.0%); while the cultivar AC Intrepid had the largest seeds and the least dockage (1.4%). This pattern was observed mainly in environments with high and medium OWBM pressure (Table 5).

Waskada, the 10% interspersed refuge in both Fieldstar VB and Unity VB, on average had significantly higher seed weight and lower dockage than these two varietal blends under all levels of midge pressure (Table 5). AC Intrepid, the 10% interspersed refuge in Goodeve VB, on average had significantly higher seed weight, but similar dockage, than Goodeve VB in all levels of midge pressure.

CONCLUSION As a group, the wheat midge-resistant varietal blends used in this study significantly out-yielded the midge-susceptible cultivars, with which they were compared, in all environments. Under high orange wheat blossom midge pressure, an 11% yield advantage, independent of other gains in yield potential, was considered directly derived from their resistance to this insect. Of the varietal blends, Shaw VB and Unity VB showed best yielding ability, followed by Fieldstar VB, while Goodeve VB tended to be earlier maturing and had significantly shorter and stronger straw. Other than yield and probably dockage, the Sm1 gene was not associated with any agronomic performance traits.

ACKNOWLEDGEMENTS

Financial support from Agriculture Development Fund is gratefully acknowledged. The authors also wish to thank Ryan Dyck, Wes Dyck, Jeff Hovland, Dale Kern, Myron Knelsen, Glenn Moskal, Richard Svistovski and Orland Thompson for technical support.

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REFERENCES Barker, P. S. 1984. Distribution of wheat midge damage on wheat in Manitoba in 1984. Proc. Entomol. Soc. Man. 40: 25-29 Barker, P. S. and McKenzie, R. I. H. 1996. Possible sources of resistance to the wheat midge in wheat. Can. J. Plant Sci. 76: 689-695. Campbell, A. B. and Czarnecki, E. 1987. Katepwa hard red spring wheat. Can. J. Plant Sci. 67: 229-230. Carrière, Y., Ellers-Kirk, C., Kumar, K., Heuberger, S., Whitlow, M., Antilla, L., Dennehy, T. J., and Tabashnik, B. E. 2005. Long-term evaluation of compliance with refuge requirements for Bt cotton. Pest Manag. Sci. 61: 327-330. Canadian Food Inspection Agency (CFIA). 2011. Shaw. [Online] Available: http://www.inspection.gc.ca/english/plaveg/pbrpov/cropreport/whe/app00007657e.shtml [7 Dec.2011]. DePauw, R. M., Clarke, J. M., Knox, R. E., Fernandez, M. R., McCaig, T. N. and McLeod, J. G. 1999. AC Intrepid hard red spring wheat. Can. J. Plant. Sci. 79: 375-378. DePauw, R. M., Knox, R. E., Thomas, J. B., Smith, M., Clarke, J. M., Clarke, F. R. and McCaig, T. N. 2009. Goodeve hard red spring wheat. Can. J. Plant. Sci. 89: 937-944. Felt, E. P. 1921. Wheat midge, Thecodiplosis mosellana (Gehin). Bull. N. Y. St. Mus. 231: 35-54. Fletcher J. 1902. Experimental farms reports for 1901. Page 212 in Report No. 16. Government of Canada. Ottawa ON. Foster, J. E., Ohm, H. W., Patterson, F. L. and Taylor, P. L. 1991. Effectiveness of deploying single gene resistance in wheat for controlling damage by the Hessian fly (Diptera: Cecidomyiidae) Environ. Entomol. 20: 964-969. Fox, S. L., McKenzie, R. I. H., Lamb, R. J., Wise, I. L., Smith, M. A. H., Humphreys, D. G., Brown, P. D., Townley-Smith, T. F., McCallum, B. D., Fetch, T. G., Menzies, J. G., Gilbert, J. A., Fernandez, M. R., Despins, T., Lukow, O. and Niziol, D. 2010. Unity hard red spring wheat. Can. J. Plant Sci. 90: 71-78. Fox, S.L., McKenzie, R.I.H., Lamb, R.J., Wise, I.L., Smith, M.A.H., Humphreys, D.G., Brown, P.D., Townley-Smith, T.F., McCallum, B.D., Fetch, T.G., Menzies, J.G., Gilbert, J.A. Fernandez, M.R., Despins, T., Lukow, O. and Niziol, D. 2012. Fieldstar hard red spring wheat. Plant Var. J. x:xx-xx. doi: 10.3198/jpr2011.06.0329crc. Fox, S. L., Thomas, J. B., Wise, I. L., Smith, M. A. H., Humphreys, D. G., Brown, P. D., Townley-Smith, T. F., McCallum, B. D., Fetch, T. G., Menzies, J. G., Gilbert, J. A., Fernandez, M. R., Despins, T. and Niziol, D. 2009. Waskada hard red spring wheat. Can. J. Plant Sci. 89: 71-78. Gallun, R. L. 1978. Genetics of biotypes B and C of the Hessian fly. Ann. Entomol. Soc. Am. 71: 481-486. Hughes, G. R. and Hucl, P. 1993. CDC Teal hard red spring wheat. Can. J. Plant Sci. 73: 193-197. Iqbal, M., Navabi, A., Salmon, D. F., Yang, R. C., Spaner, D. 2007. Simultaneous selection for early maturity, increased grain yield and elevated grain protein content in spring wheat. Plant Breeding 126: 244-250.

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Lamb, R. J., Tucker, J. R., Wise, I. L. and Smith, M. A. H. 2000. Trophic interaction between Sitodiplosis mosellana (Diptera: Cecidomyiidae) and spring wheat: Implications for yield and seed quality. Can. Entomol. 132: 607-625. Lamb, R. J., Wise, I. L., Smith, A. H., McKenzie, R. I. H. and Thomas J. 2002. Oviposition deterrence against Sitodiplosis mosellana (Diptera: Cecidomyiidae) in spring wheat. Can. Entomol. 134: 85-97. Littell, R. C., Milliken, G. A., Stroup, W. W. and Wolfinger, R. D. 1996. SAS® system for Mixed Models. SAS Institute, Inc., Cary. NC. 633 pp. McKenzie, R. I. H., Lamb, R. J., Aung, T., Wise, I. L., Barker, P. and Olfert, O. O. 2002. Inheritance of resistance to wheat midge, Sitodiplosis mosellana, in spring wheat. Plant Breeding 121: 383-388. Olfert, O. O., Mukerji, M. K. and Doane, D. F. 1985. Relationships between infestation levels and yield loss caused by wheat midge, Sitodiplosis mosellana (Gehin) (Diptera: Cecidomyiidae), in spring wheat in Saskatchewan. Can. Entomol. 117: 593-598. Rawlings, J. O. 1988. Applied regression analysis: A research tool. Wadsworth and Brooks Inc., Belmont, CA. 553 pp. Smith, M. A. H., Lamb, R. J., Wise, I. L. and Olfert, O. O. 2004. An interspersed refuge for Sitodiplosis mosellana (Diptera: Cecidomyiidae) and a biocontrol agent Macrogenes penetrans (hymenoptera: Pteromalidae) to manage crop resistance in wheat. Bulletin of Entomological Research 94: 179-188. Smith, M. A. H , Wise, I. L. and Lamb, R. J. 2007. Survival of Sitodiplosis mosellana (Diptera: Cecidomyiidae) on wheat (Poaceae) with antibiosis resistance: implication for the evolution of virulence. Can. Entomol. 139: 133-140. Tabashnik, B. E., Gassmann, A. J. Crowder, D. W., and Carrière, Y. 2008. Insect resistance to Bt crops: evidence versus theory. Nat. Biotech. 26: 199-202. Thomas, J., Fineberg, N., Penner, G., McCartney, C., Aung, T., Wise, I. and McCallum, B. 2005. Chromosome location and markers of Sm1: a gene of wheat that conditions antibiotic resistance to orange wheat blossom midge. Molecular Breeding 15: 183-192. Western Grain Research Foundation (WGRF). 2001. Endeavers and Acclamations, New wheat varieties by class. [Online] Available: www.westerngrains.com [2011 Dec 14]. Wright, A. T. and Doane, J. 1987. Wheat midge infestation of spring cereals in northeastern Saskatchewan. Can. J. Plant Sci. 67:117-120.

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Table 2. Environments (year-locations) categorized into three orange wheat blossom midge pressure groups by percentage of damaged seed. Locations 2007 2008 2009 2010 High midge pressure (%) Brandon 18.5 10.0 Indian Head 35.0 5.9 Melfort 6.5 6.5 Regina 17.6 12.3 Saskatoon 12.1 5.5 Lethbridge 8.0 Lacombe 15.7 Moderate midge pressure (%) Indian Head 3.3 4.4 Melfort 3.6 Saskatoon 4.1 5.2 Swift Current 4.2 3.2 2.7 Low midge pressure (%) Brandon 2.6 0.9 Melfort 1.0 Regina 2.0 0.5 Swift Current 0.9 Lethbridge 0.1 0.0 0.5 Lacombe 1.7 0.8 0.1

11 Table 1. Coordinates, plots size, seeding date and soil fertilization of orange wheat blosom midge trials grown in four consecutive years at eight locations in western Canada. Plot sizez Seeding date N - P Fertilizationy (kg ha-1) Locations Coordinates (m2) 2007 2008 2009 2010 2007 2008 2009 2010 Brandon 50.0º N, 99.9º W 3.7 May 28 May 20 May 14 May 19 172 - 35 213 - 24 195 - 44 162 - 29 Indian Head 50.5º N, 103.7º W 2.8 May 28 May 14 May 19 May 14 50 - 47 111 - 65 111 - 52 174 - 23 Melfort 52.8º N, 104.6º W 3.7 May 18 May 16 May 13 May 16 114 - 70 131 - 70 140 - 70 108 - 44 Regina 50.5º N, 104.6º W 2.7 May 17 May 20 May 26 May 17 255 - 69 168 - 67 125 - 67 151 - 73 Saskatoon 52.1º N, 106.7º W 3.0 June 05 May 30 May 30 June 13 96 - 39 104 - 66 221 - 139 185 - 102 Swift Current 50.3º N, 107.7º W 2.7 May 25 May 13 May 11 May 17 124 - 50 104 - 55 106 - 56 125 - 67 Lethbridge 49.7º N, 112.8º W 2.8 May 17 May 18 May 22 May 16 275 - 63 192 - 92 145 - 89 167 - 78 Lacombe 52.5º N, 113.7º W 2.7 May 18 May 04 May 12 May 20 128 - 63 131 - 51 120 - 60 223 - 76 z Harvested area y Soil test + additional nutrients. Nitrogen (N) was measured in the 0-60 cm soil profile, while phosphorus (P) was measured in the 0-15 cm soil profile Table 3. Growing season (May-Sep) precipitation and growing degree days for the period 2007-2010, compared to the long-term average (normal), at eight locations in western Canada. Precipitation (mm) Growing Degree Days (ºC)z Locations 2007 2008 2009 2010 Averagey Normalx 2007 2008 2009 2010 Averagey Normalx Brandon 283 354 272 369 320 318 1664 1509 1550 1568 1573 1610 Indian Head 272 244 287 372 294 296 1500 1371 1374 1394 1410 1550 Regina 202 248 184 388 256 259 1620 1443 1450 1421 1484 1541 Melfort 298 190 260 392 285 284 1404 1375 1352 1338 1367 1424 Saskatoon 293 196 272 475 309 240 1494 1467 1450 1398 1452 1523 Swift Current 124 330 177 499 283 238 1681 1475 1549 1351 1514 1534 Lethbridge 167 387 241 394 297 224 1752 1572 1610 1415 1487 1545 Lacombe 477 300 232 514 381 339 1296 1234 1246 985 1190 1207 Average 228 253 207 425 303 275 1551 1431 1448 1359 1447 1492 z Base temperature = 5oC y Growing season four-year (2007-2010) average x Growing season long-term (1971-2000) average

12 Table 4. Variances of random factors and F-tests of fixed factors from combined analysis with PROC MIXED of heading, maturity, plant height, seed yield, seed weight and dockage of eight wheat cultivars (four midge-resistant and four midge-susceptible), grown during four consecutive years at eight locations in western Canada. Environments (year-locations) were grouped into three levels of orange wheat blossom midge pressure. Effect Heading Maturity Plant height Seed yield Seed weight Dockage High midge pressure (Variancesz) Environment 22.89 * 134.62 * 132.60 ** 914255 * 7.93 * 1.93 * Rep (environment) 0.22 * 1.58 * 7.33 *** 42198 *** 0.20 ** 0.00 Environment x cultivar 0.95 *** 0.81 ** 3.61 *** 47479 *** 1.58 *** 0.32 *** Residual 0.61 *** 0.41 *** 10.27 *** 55697 *** 0.73 *** 0.14 *** Cultivar (F-test) 10.37 *** 6.49 *** 16.56 *** 20.45 *** 22.99 *** 15.53 *** Moderate midge pressure (Variances) Environment 13.72 61.35 172.03 * 1767981 * 36.02 * 25.10 * Rep (environment) 0.04 0.57 * 5.19 ** 20950 ** 0.12 0.21 ** Environment x cultivar 0.58 *** 0.96 ** 4.69 *** 23146 *** 1.18 *** 1.45 *** Residual 0.30 *** 2.60 *** 7.63 *** 30397 *** 1.21 *** 0.24 *** Cultivar (F-test) 6.50 *** 5.04 *** 11.65 *** 8.86 *** 18.34 *** 2.31 * Low midge pressure (Variances) Environment 13.07 * 34.00 * 180.46 * 3014761 * 11.00 * 1.75 * Rep (environment) 0.48 ** 1.27 *** 11.08 ** 84673 *** 0.32 ** 0.08 *** Environment x cultivar 0.42 *** 1.38 *** 8.09 *** 101116 *** 0.91 *** 0.13 *** Residual 0.44 *** 2.93 *** 10.58 *** 75072 *** 1.08 *** 0.07 *** Cultivar (F-test) 10.32 *** 7.42 *** 6.21 *** 5.03 *** 40.19 *** 9.50 *** z Z-test for significant variances (variances are greater than zero); * P < 0.05, ** P < 0.01, *** P < 0.001.

13 Table 5. Heading, maturity, plant height, seed yield, seed weight and dockage of four midge-resistant wheat varietal blends (VB, carrying the Sm1 gene) and four midge-susceptible wheat cultivars (not carrying the Sm1 gene), grown during four consecutive years at eight locations in western Canada. Environments (year-locations) were grouped into three levels of orange wheat blossom midge pressure.

Heading Maturity Plant height Seed yield Seed weight Dockage Treatments and Sm1 contrast (days)z (days)z (cm) (kg ha-1) (mg seed-1) (%) High midge pressure (12.8% seed damage) Fieldstar VB 53.4 cdy 99.5 ab 104.0 a 3606 b 30.2 e 3.50 a Goodeve VB 53.0 d 98.4 bc 96.7 d 3374 c 33.4 bc 1.72 cd Shaw VB 54.6 a 100.3 a 104.3 a 3905 a 33.9 b 1.71 cd Unity VB 53.2 d 100.0 a 101.2 b 3759 ab 32.0 d 2.39 b AC Intrepid 51.3 e 97.3 c 98.9 c 3299 cd 36.6 a 1.38 d CDC Teal 54.2 bc 99.9 a 98.0 cd 3170 d 32.7 cd 1.73 cd Katepwa 54.4 ab 98.6 b 103.0 ab 2903 e 31.9 d 1.90 c Waskada 52.7 d 100.2 a 103.1 ab 3380 c 33.4 bc 1.58 cd SEDx 0.482 0.592 1.02 101 0.542 0.243 Moderate midge pressure (3.8% seed damage) Fieldstar VB 57.6 ab 105.9 bc 98.2 ab 3659 bc 31.4 e 3.77 a Goodeve VB 56.6 b 105.3 bc 91.6 d 3532 cd 35.5 b 1.78 b Shaw VB 58.1 a 106.3 ab 99.4 a 3924 a 34.6 bc 2.66 a Unity VB 57.1 ab 106.2 ab 96.2 bc 3898 a 33.6 cd 2.61 ab AC Intrepid 55.3 c 103.7 d 93.9 cd 3614 bc 37.4 a 1.62 b CDC Teal 57.7 a 105.8 bc 94.2 cd 3501 cd 32.9 d 2.64 cde Katepwa 57.9 a 104.7 cd 99.6 a 3403 d 33.0 d 2.75 cd Waskada 56.6 bc 107.6 a 99.3 a 3716 b 35.1 b 2.39 de SED 0.513 0.731 1.29 88w 0.610w 0.614w Low midge pressure (0.9% seed damage) Fieldstar VB 55.9 bc 106.2 ab 98.1 bcd 4782 bc 33.4 e 2.11 a Goodeve VB 55.4 c 105.2 bc 95.0 e 4712 c 37.7 b 1.18 c Shaw VB 57.1 a 106.3 ab 101.3 a 5054 ab 37.2 bc 1.37 bc Unity VB 56.0 bc 106.4 ab 98.8 abc 5120 a 35.4 d 1.53 b AC Intrepid 54.1 d 103.5 d 96.1 de 4837 bc 39.8 a 1.17 c CDC Teal 56.1 bc 105.7 bc 96.3 cde 4778 c 36.4 c 1.14 c Katepwa 56.5 ab 104.9 c 100.9 a 4390 d 35.1 d 1.34 bc Waskada 55.4 c 107.4 a 100.0 ab 4903 abc 37.9 b 1.06 c SED 0.390 0.620 1.34 141 0.443 0.155 z Heading and maturity are expressed as days from seeding y Means followed by same letters do not differ significantly based on t-test at P ≤ 0.05. x Standard error of the difference between treatment means. w Average SED was used when there were missing plot values in the analyses of variance.

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Table 6. Comparison of antibiotic (Sm1) versus non antibiotic (Non Sm1) cultivars, and comparison of antibiotic and antixenotic (Waskada) cultivars versus fully midge susceptible (Susceptibles) cultivars for heading, maturity, plant height, seed yield, seed weight and dockage.

Heading Maturity Plant height Seed yield Seed weight Dockage Group of treatments and Contrasts (days)z %y (days)z % (cm) % (kg ha-1) % (mg seed-1) % (%) %

High midge pressure (12.8% seed damage) Sm1 cultivars 53.6 99.5 101.5 3661 32.4 2.33 Non Sm1 cultivars 53.2 99.0 100.8 3188 33.6 1.65 Sm1 + Waskada cultivars 53.4 99.7 101.9 3605 32.6 2.18 Susceptible cultivars 53.3 98.6 101.0 3124 33.7 1.67 Sm1 vs. Non Sm1x 0.389 0.73 0.528 0.53 0.760 *** 0.75 473 *** 14.8 -1.279 *** -3.80 0.681 *** 41.3 SEDw 0.241 0.296 0.507 50.6 0.271 0.122 Sm1 + Waskada vs. Susceptiblesx 0.078 0.15 1.083 *** 1.10 1.861 *** 1.86 481 *** 15.4 -1.173 *** -3.48 0.509 *** 30.5 SEDw 0.249 0.306 0.524 52.2 0.280 0.126 Moderate midge pressure (3.8% seed damage) Sm1 cultivars 57.3 105.9 96.4 3753 33.8 2.71 Non Sm1 cultivars 56.9 105.5 96.8 3558 34.6 2.35 Sm1 + Waskada cultivars 57.2 106.3 100.0 3746 34.0 2.64 Susceptible cultivars 57.0 104.7 96.0 3506 34.4 2.34 Sm1 vs. Non Sm1 0.463 0.81 0.479 0.45 -0.469 0.48 195 *** 5.47 -0.845 *** -2.44 0.356 15.2 SED 0.257 0.366 0.642 43.9 0.305 0.307 Sm1 + Waskada vs. Susceptibles 0.210 0.37 1.544 *** 1.47 0.967 1.01 240 *** 6.84 -0.428 ** -1.24 0.303 13.0 SED 0.265 0.378 0.663 45.3 0.315 0.317 Low midge pressure (0.9% seed damage) Sm1 cultivars 56.1 106.1 98.3 4917 35.9 1.55 Non Sm1 cultivars 55.6 105.4 98.3 4727 37.3 1.18 Sm1 + Waskada cultivars 56.0 106.3 98.6 4914 36.3 1.45 Susceptible cultivars 55.6 104.7 97.8 4669 37.1 1.22 Sm1 vs. Non Sm1 0.571 ** 1.03 0.693 * 0.66 -0.067 0.07 190 ** 4.01 -1.379 *** -3.70 0.365 *** 30.9 SED 0.195 0.310 0.669 70.7 0.222 0.077 Sm1 + Waskada vs. Susceptibles 0.391 0.70 1.636 *** 1.56 0.843 0.86 246 ** 5.26 -0.759 ** -2.05 0.229 ** 18.8 SED 0.201 0.320 0.691 73.0 0.229 0.080 z Heading and maturity are expressed as days from seeding. y Difference between the two groups of treatments being compared in the contrasts. x Contrast estimates and significance; * P < 0.05, ** P < 0.01, *** P < 0.001. w Standard error of the difference for the contrasts.

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Midge 2011 ADF20070092 Final Report · 2018-10-23 · 3 Treatments were 4 resistant wheat variety blends (BW362 VB, BW365 VB, BW394 VB, BW841 VB) and 4 susceptible wheat varieties