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a Dep. of Crop and Soil Sciences, Washington State Univ., Pullman, WA, 99164
b Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59715
c Western Triangle Research Center, P.O. Box 974, Conrad, MT 59425
* Corresponding author (bruckner{at}montana.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: WSMV, Wheat streak mosaic virus • ELISA, enzyme-linked immunosorbent assay • PCR, polymerase chain reaction
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The WSMV-resistant germplasm, KS93WGRC27, contains a translocation from Thinopyrum intermedium (Host) Barkworth and Dewey chromosomal arm 4Js that carries the resistance gene Wsm1 (Lay et al., 1971; Friebe et al., 1991; Gill et al., 1995; Chen et al., 1998). The line is a BC3F2-derived line from the backcross of the high yielding hard red winter wheat cultivar Karl with CItr 17884 (Gill et al., 1995). CItr 17884 is a ‘Centurk’ backcross derivative translocation line carrying Wsm1 and with good bread baking properties (Wells et al., 1982). Karl has excellent grain protein content, grain milling, and baking quality (Sears et al., 1991). Because of the limited size of the alien gene translocation, as detected by in situ hybridization, the lack of virus accumulation, and the acceptable quality of parental lines, KS93WGRC27 shows promise as a reliable source of resistance to WSMV in a background with acceptable end-use quality.
The Montana spring wheat breeding program developed advanced spring wheat progeny with and without the 4Js translocation using a polymerase chain reaction (PCR) primer specific to the T. intermedium segment carrying the Wsm1 gene (Talbert et al., 1996). These progeny were suitable for evaluating the overall impact of the T. intermedium segment on WSMV resistance and agronomic and end use quality. Evaluating the effects of the translocation from T. intermedium when incorporated into cultivated wheat is important to the development of productive, WSMV-resistant wheat cultivars. Knowledge gained from experiments with spring wheat may serve as a template for parallel applications in winter wheat.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Primer Evaluation
Total genomic DNA was extracted following the procedures of Lassner et al. (1989) from five individual plants for each entry. Approximately 200 ng of DNA was used per PCR reaction. The protocol for thermocycling was an initial denaturation of the DNA at 94°C for 4 min, followed by 29 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min and 20 s. A final 7 min at 72°C was followed by an infinite hold at 4°C. A PCR product from an entry carrying the T. intermedium translocation produces a band of approximately 500 base pairs thus identifying genotypes containing the Wsm1 gene (Talbert et al., 1996). On the basis of PCR evaluation a total of 22 translocation positive (resistant) and 36 translocation negative (susceptible) lines (7R and 13S, 5R and 5S, 5R and 11S, and 5R and 7S, in Populations 1–4, respectively) were advanced for further evaluation.
Field Trial Evaluations
Field trials were conducted in 1998 and 1999 at the Western Triangle Agricultural Research Center north of Conrad, MT, and at the Post Field Research Farm at Bozeman, MT. The experiment was planted in split plot design with three replications in each environment. The four populations were main plots with resistant and susceptible lines and appropriate parental lines randomized as subplots for each population. Lines were grown in four-row plots seeded at 8 g per 3.0-m row in Conrad and 9 g per 3.7-m row at Bozeman. Rows were spaced 30 cm apart with equidistant spacing between adjacent plots. In 1999, field experiments were established with F7 seed from one replication of nontreated entries grown at Bozeman in 1998. Experiments were planted 21 April and 5 May 1998 and on 15 and 16 April 1999 at Conrad and Bozeman, respectively. At Conrad, plots were harvested on 19 to 20 Aug. 1998 and on 24 Aug. 1999. At Bozeman, plots were harvested 21 Aug. 1998 and 8 Sept. 1999.
In 1998, the first row of each four-row plot was manually inoculated with WSMV by the hand rub method with plants at the two to four leaf stage (McKinney and Sando, 1951). In 1999, plots were divided between the center two rows, and the left two rows of each four-row plot were inoculated with WSMV. Inoculum was prepared from greenhouse grown McNeal wheat in 1998, and ‘Tiber’ wheat in 1999, that had been infected with the Conrad virus isolate (Carroll et al., 1982). Plant material was homogenized in a 0.1 M potassium-sodium phosphate buffer (pH 6, 
500 g tissue/L buffer) with carborundum added to facilitate viral introduction. At both 15 and 30 d after inoculation, two 7.6-cm plant sections of each inoculated row were harvested. These harvested sections were used to estimate virus accumulation by means of enzyme-linked immunosorbent assay (ELISA) (Edwards and Cooper, 1985). Polyclonal antibodies utilized for the ELISA were made from WSMV purified from an adapted protocol described by Brakke and Ball (1968).
Noninoculated rows were evaluated for heading date, plant height, physiological maturity, grain protein, test weight, and yield. Inoculated rows were evaluated for plant height, visual disease rating, ELISA, test weight, and yield. Inoculated treatments were not rated for heading date and physiological maturity because of a high degree of plant-to-plant variability within rows including extended vegetative growth and prolonged tillering. Yield reduction due to WSMV inoculation was calculated for each plot as yield reduction in inoculated rows relative to the noninoculated rows in the same plot. The grain protein content from noninoculated plots was measured with a Tecator Infratec 1225 Grain Analyzer (Foss North America, Silver Springs, MD) in the Cereal Quality Lab at Montana State University, Bozeman.
Inoculated rows were rated on a scale of 0 to 3 to measure disease response. This disease scale used 0 to indicate no symptoms indicative of WSMV, 1 to indicate mild chlorosis, 2 to indicate moderate chlorosis, and 3 to indicate severe disease response or necrosis. Inoculated leaf tissue samples were weighed (2.5–5 g) and pulverized in 50-mL conical tubes by means of a Omni International TH hand grinder (Warrenton, VA). Five mL/g of PBS-Tween solution (pH 7.4) was added to each tube, which were then vortexed for 1 min. A 1.5-mL sample was poured into a 1.5-mL Eppendorf tube and spun at 10 000 g for 45 s. The ELISA tests were performed as reported by Edwards and Cooper (1985). Absorbance was measured on a Molecular Devices Kinetic Microplate Reader (Sunnyvale, CA) at 405 nm, after a 20 min incubation with p-nitrophenyl phosphate substrate at 1 mg/mL in substrate buffer.
Cereal Quality Evaluation
End-use quality was determined only on noninoculated treatments since grain yield from inoculated treatments was too low to practically generated adequate grain volume for quality determination. Grain from one replication of noninoculated plots from Conrad and Bozeman was cleaned and evaluated for end-use qualities by approved methodology (AACC, 1995). One week prior to milling, the samples were adjusted to 120 g kg-1 moisture with a Dickey-John/Motomoco Model 919 Automatic Grain Moisture tester (Auburn, IL). Twenty-four hours before milling the wheat was tempered a second time to 155 g kg-1 moisture. Samples were milled on a Brabender Automat Mill (South Hackensack, NJ) to obtain flour, bran, and shorts/middlings. The separated flour was analyzed for flour yield, flour ash, and flour protein. Mixograph mixing time and absorption were determined for each sample. Mixograph tolerance was scored from 1 to 8 (weak to strong) by comparing mixograph curves to standard mixograph reference charts modified for protein content from Pomeranz (1987).
Baking evaluations were performed with 100-g flour loaves. In preparing the loaves, baking absorption and bake mixing time was recorded. The baking method consisted of a 90 min, sugar-based, fermented dough system. The bread formula contains 100 g flour, 6 g sugar, 3.5 g shortening, 1.8 g yeast, 1.5 g salt, 180 µg g-1 Doh-Tone (American Ingredients Co., Kansas City, MO), and 150 µg g-1 ascorbic acid as an oxidant. The finished loaves were analyzed for bake volume (canola seed displacement) and crumb grain score by a subjective 0 to 5 scale (5 = best).
Statistical Analysis
Analysis of variance was computed for each trait. The analysis was first computed for each population-environment combination. The analysis was then combined across environments for each population, and also combined over populations. The mean of resistant lines was compared with the mean of susceptible lines for each trait. Replications and lines within resistant and susceptible classes and their interactions were treated as random, while other effects were considered fixed. Tests of significance for fixed effects in the model were constructed by means of the Satterthwaite (1946) procedure to compute the appropriate error term from linear combinations of mean squares.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At Bozeman in 1999, resistant lines from Populations 1, 3, and 4 showed significantly less yield loss due to WSMV than susceptible lines. Resistant and susceptible lines from Population 2 did not differ at Bozeman in 1999. The mean yield reduction due to WSMV inoculation was less than 1% for resistant entries at Bozeman in 1999 and 13% for susceptible entries. At Conrad in 1999, only Populations 3 and 4 displayed significant differences between susceptible and resistant groups in the yield reduction due to WSMV inoculation. At Conrad in 1999, the overall yield reduction due to WSMV for resistant lines was 4% and for susceptible lines the yield reduction was 11%.
Mean test weight of resistant lines was 14.7 kg m-3 higher than mean test weight of susceptible lines (Table 1). Test weights of resistant lines were higher than test weights of susceptible lines in Populations 1 and 4 but similar in Populations 2 and 3 (Table 1). Mean test weights of resistant lines were 15.3 and 27.1 kg m-3 higher than test weights of susceptible lines in Populations 1 and 4, respectively. Test weights of susceptible and resistant groups from Population 1 differed at both locations in 1999 but neither location in 1998, although trends were similar in all environments (not shown). In Population 4, test weights of resistant and susceptible groups differed at both locations in 1998, but not 1999 (not shown). Test weight reductions due to WSMV inoculation were significantly greater in susceptible lines than resistant lines in all populations and environments (Table 1).
No significant differences were observed for height among resistant and susceptible groups in Populations 1, 3, and 4 (Table 1). Resistant lines of Population 2 were significantly taller than susceptible lines for both years at Conrad but were not significantly different in height in either year at Bozeman (data not shown). The overall height reduction of WSMV inoculated susceptible lines was significantly greater than the height reduction of resistant lines over the entire experiment by 2.8 cm (Table 1). All populations except Population 4 showed a significantly greater height reduction in susceptible lines than in resistant lines (Table 1). Population 4 resistant lines showed height reductions that were nearly the same as susceptible lines in all environments.
Resistant and susceptible lines from all populations were significantly different for ELISA readings, a qualitative estimate of virus accumulation in the plant (Table 1). There was a 3- to 10-fold difference between resistant and susceptible mean values for each population (Table 1). Consistent with ELISA data, susceptible lines showed significantly higher levels of disease symptoms than resistant lines in all populations (Table 1). Differences in visual disease ratings for resistant and susceptible groups were greatest for Populations 1 and 3 and more moderate in Populations 2 and 4.
There were no significant differences among resistant and susceptible groups for grain protein in any population or environment (Table 1). The overall mean protein levels were the same for resistant, susceptible, and parental lines at 133 g kg-1 (Table 1). The results from milling and baking tests are reported in Table 3. Statistical analysis of cereal quality data indicated no significant differences between resistant and susceptible groups except for a lower mixograph absorption for resistant lines relative to susceptible lines across populations and environments.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Yield reductions due to WSMV inoculation were 5 and 32% for resistant and susceptible groups, respectively (Table 1). This indicates that Wsm1 is an effective source of WSMV resistance. The small yield reduction due to inoculation reported for resistant lines (0.8–8.5%) may have been due to a small level of heterogeneity in some resistant lines being tested. Regardless, the yield performance of resistant lines exceeded that of susceptible and parental lines under inoculated conditions for all populations in all environments.
Test weights of resistant lines were significantly higher than test weights of susceptible lines and higher than parental lines (Table 1). This effect was most pronounced in Populations 1 and 4 (Table 1). Higher test weights are associated with flag leaf health during the grain fill period and correlated to higher flour extraction potential. Although not determined in this study, the improved test weight resulting from the alien chromosome segment may have resulted from additional unidentified disease resistance or abiotic stress tolerance factors or from modification of yield components such as fewer kernels per spike or decreased tiller density. The effectiveness of the resistance gene, Wsm1, to minimize test weight reductions due to WSMV infection was readily apparent in all populations (Table 1). The average test weight reduction for resistant lines was 17.3 kg m-3. The average test weight reduction due to WSMV for susceptible lines was 73.4 and 102.0 kg m-3 for the parental lines (Table 1).
Decreased height reduction for inoculated resistant lines reported here supports previous yield and test weight results showing the effectiveness of the resistance conferred by the Wsm1 gene. The minimal height reduction that was seen in resistant inoculated lines may be simply the result of stress due to the abrasion of young leaf tissue when inoculum was introduced. Under conditions in which WSMV was more severe, stunting would likely be more severe in susceptible lines, leading to a greater height reduction in response to WSMV inoculation. The T. intermedium translocation was also found to have minimal effects on heading date (on average, resistant lines 1 d later than susceptible lines) and no effect on physiological maturity (data not shown).
Visual symptoms or disease ratings are not always accurate in assessing the level of WSMV replication. However, in this experiment, symptom expression and ELISA were consistent, indicating a large decrease in visual WSMV symptoms and ELISA readings in resistant lines relative to susceptible lines. ELISA and WSMV symptomology were also consistent with data from inoculated treatments showing minimal yield, test weight, and plant height reductions in resistant germplasm. The Wsm1 resistance gene effectively limits virus accumulation, reduces disease symptoms, and reduces yield loss to WSMV.
Symptom expression and yield reductions due to WSMV inoculation were far less at both locations in 1999 than reported for 1998 (Table 2), despite the fact that mean ELISA absorbance was as high or higher in 1999 than in 1998 in all populations and at both locations (data not shown). In all environments and populations, resistant lines had significantly lower mean ELISA absorbance than susceptible lines, ranging from a 2.3- to 56-fold difference (average 13.3-fold difference) for mean ELISA absorbance, depending on population and environment. This indicates the virus accumulated and had differential effects in resistant and susceptible classes in all environments. Decreased rainfall and higher mean temperatures at both locations in 1999 may have resulted in environmental conditions less conducive to severe symptom expression and yield loss. Similar ELISA absorbance occurred both years but differential symptom expression and response was seen at the whole plant level. This inconsistency and unpredictability of the consequences of WSMV infection from year to year and environment to environment reinforces the case for development of yield-competitive, WSMV-resistant varieties which do not exhibit a yield penalty in the absence of disease.
Average whole grain protein content of resistant lines was the same as the susceptible and parental lines (Table 1). Protein content of wheat with alien chromosome disease resistance genes has long been a short coming in developing varieties for agricultural production (Wells et al., 1982). It appears here that the presence of an alien chromosome segment in resistant lines had no detrimental effect on grain protein content.
Incorporation of the T. intermedium translocation carrying Wsm1 into adapted, high-quality Montana spring wheat germplasm had no detrimental effects on end-use quality parameters (Table 3). With exception of mixograph absorption, WSMV-resistant and WSMV-susceptible groups were similar for all milling and baking parameters. Mean mixograph absorption was slightly lower in resistant lines than susceptible lines. Milling and baking qualities of both resistant and susceptible lines were similar to parental lines and acceptable by industry standards.
Previous studies evaluating chromosomal translocations between wheat and rye have associated the translocation with increased grain yield and decreased end-use quality in wheat (Espititia-Rangel et al., 1999). However, studies evaluating the 1AL.1RS and 1BL.1RS translocation from rye have shown that many of these detrimental effects on end-use quality can be decreased by rigorous selection of the background genotype (Carver and Rayburn, 1995; Lee et al., 1995). Yield loss was shown to be associated with WSMV resistance in this study, but such losses should be overcome by selection as suggested by the range of resistant entry yields. These results indicate a highly effective source of WSMV resistance with potentially positive effects on grain test weight and no detrimental effects on end-use quality. The resistance to WSMV present in KS93WGRC27, associated with a Thinopyrum intermedium translocation, shows that it is possible to have alien gene expression without a major penalty in agronomic and end-use quality.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication January 2, 2001.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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