Plot-to-plot, row-to-row and plant-to-plant outcrossing studies in oilseed rape

Transcription

1 Plot-to-plot, row-to-row and plant-to-plant outcrossing studies in oilseed rape J. L. Cuthbert 1, and P. B. E. McVetty 2 1 Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg, Manitoba, Canada R3T 2M9; 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Received 5 April 2001, accepted 20 June Cuthbert, J. L. and McVetty, P. B. E Plot-to-plot, row-to-row and plant-to-plant outcrossing studies in oilseed rape. Can. J. Plant Sci. 81: Unidirectional outcrossing rates were assessed between neighboring plots, rows (spaced 40, 80 and 120 cm apart) and plants of oilseed rape (Brassica napus L.) grown in simulated plant breeding field trials, using the transgenic dominant bromoxynil herbicide resistance gene as a marker. Bromoxynil susceptible (pollen recipient) plots, rows and plants were planted in the field and surrounded by bromoxynil-resistant plots, rows and plants, respectively. The field trials were conducted at Winnipeg, Carman, and Portage la Prairie, Manitoba, in and. Seed produced on the susceptible plots and rows was harvested and then planted in the field the following year with all emerged seedlings sprayed at 750 g a.i. ha 1 bromoxynil to identify resistant individuals. Approximately seedlings were screened in and 1998 with resistant individuals identified, each resistant individual being the result of an outcrossing event. The overall mean plot-to-plot outcrossing rate was 4.0% (± 0.23). The overall mean row-to-row outcrossing rate was 9.5% (± 0.62) for the 40-cm row spacing, 5.6% (± 0.37) for the 80-cm row spacing and 3.9% (± 0.25) for the 120-cm row spacing. For the plant-to-plant trials, seed produced on the susceptible plants was harvested and then a sample was planted in the greenhouse with all emerged seedlings sprayed at 560 g a.i. ha 1 bromoxynil to identify resistant individuals. The overall mean plant-to-plant outcrossing rate was 21.0% (± 1.73). Outcrossing rates of this magnitude have significant implications for all oilseed rape breeding programs. Methods to minimize outcrossing are discussed. Key words: Brassica napus L., outcrossing rates, transgenic dominant bromoxynil resistance marker Cuthbert, J. L. et McVetty, P. B. E Hybridation du colza par croisement extérieur parcelle/parcelle, rang/rang et plant/plant. Can. J. Plant Sci. 81: Les auteurs ont évalué le taux de croisement extérieur unidirectionnel entre des parcelles, des rangs (espacés de 40, de 80 et de 120 cm) et des plants de colza (Brassica napus L.) cultivés lors d un simulacre d essais d hybridation sur le terrain. Les chercheurs ont utilisé le gène dominant de résistance au bromoxynil, un désherbant, comme marqueur. Les parcelles, les rangs et les plants de colza sensible au bromoxynil (à polliniser) ont respectivement été entourés de parcelles, de rangs et de plants de colza résistant sur le terrain. Les essais se sont déroulés à Winnipeg, à Carman et à Portage la Prairie, au Manitoba, en et. Les graines issues des parcelles et des rangs de colza sensible ont été récoltées puis plantées au champ l année suivante et les plantules ont été arrosées de 750 g de bromoxynil par hectare de façon à permettre l identification des plants résistants. Environ plantules ont subi ce traitement en et 1998, ce qui a entraîné l identification de sujets résistants, chacun provenant d un croisement extérieur. Le taux moyen de croisement extérieur entre parcelles s élevait à 4,0 % (± 0,23), contre 9,5 % (± 0,62) entre les rangs espacés de 40 cm, 5,36 % (± 0,37) entre ceux espacés de 80 cm et 3,9 % (± 0,25) entre ceux espacés de 120 cm. On a recueilli les graines issues des essais d hybridation entre plants sur les sujets sensibles avant d en semer un échantillon en serre et de pulvériser sur les plantules 560 g de bromoxynil par hectare, toujours pour identifier les sujets résistants. Le taux de croisement extérieur moyen s établissait à 21,0 % (± 1,73). Un taux aussi haut aura d importantes répercussions sur les programmes d hybridation du colza. Suit une discussion sur les moyens permettant de réduire au minimum les croisements extérieurs. Mots clés: Brassica napus L., taux de croisement extérieur, marqueur dominant transgénique de la résistance au bromoxynil The breeding system of a crop, (self, intermediate or cross-pollinated), determines in part the methods a plant breeder uses to develop new cultivars. Oilseed rape (Brassica napus L.) is a predominately self-pollinating crop (Olsson 1960) with sticky, entomophilous pollen well suited to transfer by insects (Eisikowitch 1981). Oilseed rape exhibits an average plant-to-plant outcrossing rate of 20 to 40% in Europe (Becker et al. 1992), and a range of 5 to 75% outcrossing in Canada (Lewis and Woods 1991). These results indicate that oilseed rape can readily outcross within its species, which can lead to 657 contamination of breeding lines and/or unexpected genetic segregation in a field based breeding program. Despite the outcrossing rates reported in the literature, oilseed rape breeders predominately use breeding methods appropriate for self-pollinating crops. The consequences of using a self-pollinated crop breeding approach on the genetic purity of oilseed rape strains are largely unknown. Prior to the incorporation of novel herbicide resistance into oilseed rape, phenotypic markers such as erucic acid in the seed oil or petal color variants were used to assess outcrossing rates. These markers to monitor outcrossing rates

2 658 CANADIAN JOURNAL OF PLANT SCIENCE are less than ideal since it is difficult to analyze large numbers of samples for erucic acid, leading to large standard errors of estimated outcrossing rates, while petal color is known to affect pollinator plant preferences which could bias the estimation of outcrossing rates. In contrast, the use of dominant transgene controlled novel herbicide resistance in oilseed rape provides an easy and accurate way to assess outcrossing rates. Therefore, the objectives of this research were to determine 1) plot-to-plot outcrossing rates, 2) row-to-row outcrossing rates for row spacings of 40, 80 and 120 cm, and 3) plant-to-plant outcrossing rates, with all oilseed rape grown in typical plant breeding field layouts, using transgenic dominant bromoxynil herbicide resistance as a marker. MATERIALS AND METHODS Outcrossing Rate Field Trials Outcrossing rate field trials were established at the University of Manitoba Research Farm at Carman, Manitoba (hereafter Carman ), at the Integrated Crop Management Services Research Farm at Portage la Prairie, Manitoba (hereafter Portage ), and at the University of Manitoba at Winnipeg, Manitoba (hereafter Winnipeg ) in and. Near-isogenic populations were used in three separate trial designs to determine the outcrossing rates in typical oilseed rape breeding field layouts. A University of Manitoba bred oilseed rape population designated as 3801 was used as the susceptible or pollen recipient, while the pure breeding bromoxynil resistant BC 4 F 4 population of the cross , with used as the recurrent parent, was used as the resistant or pollen donor. The near-isogenic populations differed primarily for the bromoxynil resistance gene. The agronomic, phenological and phenotypic traits for the near-isogenic populations were virtually identical. A small-plot belt cone seeder was used to seed the trials at a rate of 8 kg ha 1. Carbofuran (10% granules) insecticide was banded with the seed at a rate of 12 kg a.i. ha 1 to control flea beetles (Phyllotreta cruciferae Goeze and P. striolata F.). Carman was seeded on 23 May and 15 May ; Portage was seeded on 30 May and 27 May and Winnipeg was seeded on 28 May and 26 May. These seeding dates are all well before the Manitoba Crop Insurance planting date deadline of 10 June for Brassica napus seeded in southern Manitoba (Manitoba Agriculture 2001). Plot-to-plot Outcrossing Trials These trials consisted of individual six-row susceptible plots completely surrounded by six-row bromoxynil resistant plots. Eight replicates were planted at each location in each year. Each plot was 3 m long and 1.2 m wide and consisted of six rows of oilseed rape spaced 20 cm apart. Plots were spaced 60 cm apart in the ranges. The trials were seeded over eight ranges with 1.5-m-wide pathway between ranges. The bromoxynil-susceptible plots were hand-cut at maturity, placed in jute bags, air dried for up to 4 wk and then threshed using a meticulously cleaned stationary thresher. Seed samples from the harvested plots were screened for bromoxynil resistance in the field the following summer. Row-to-row Outcrossing Trials These trials consisted of bromoxynil-susceptible single rows bordered on both sides by bromoxynil-resistant rows. The single 3-m-long rows were planted using three different row spacings (40, 80 and 120 cm). Eight replicates of each row spacing were planted at each location in each year. The trials were seeded over eight ranges with 1.5-m-wide pathway between ranges. Due to field space constraints, the 120-cm row spacing treatment was not planted at Carman in. The bromoxynil-susceptible rows were hand-cut at maturity, placed in jute bags, air dried for up to 4 wk and then threshed using a meticulously cleaned stationary thresher. Seed samples from the harvested rows were screened for bromoxynil resistance in the field the following summer. Plant-to-plant Outcrossing Trials These trials consisted of one bromoxynil-susceptible plant grown in the center of a six-row bromoxynil resistant plot. Each plot was 3 m long and 1.2 m wide consisting of six rows of bromoxynil-resistant oilseed rape spaced 20 cm apart. Plots were spaced 60 cm apart in the ranges. The trial was seeded over eight ranges with 1.5-m-wide pathway between ranges. Bromoxynil-susceptible plants, phenologically synchronized with plant development in the plots, were transplanted between row 3 and 4 of the six-row plot at the two- to four-leaf stages at the Winnipeg location only in. In, plant-to-plant trials were grown at Carman, Portage, and Winnipeg with 10 bromoxynil-susceptible seeds hand-planted between row 3 and 4 of the sixrow plot immediately after the bromoxynil-resistant plots were seeded. The number of bromoxynil-susceptible plants was thinned so only one bromoxynil-susceptible plant remained in each plot at flowering. Sixteen replicates were used at all planted locations in both years. An identification tag was placed on each bromoxynil-susceptible plant so it could be easily identified. At maturity, all bromoxynil-susceptible plants were removed and placed in paper bags to dry. The dried bromoxynil-susceptible plants were hand threshed and a sample of the seed from each plant was screened indoors for bromoxynil resistance. Assessing Plot-to-plot and Row-to-row Outcrossing Rates Outdoor screening trials, to determine outcrossing rates for plot-to-plot and row-to-row treatments, were conducted in the field at Winnipeg and Carman in, and at Carman in The screening trials in were planted on 30 May at Winnipeg and 18 June at Carman. In 1998, the screening trials at Carman were planted on 12 May and 27 May. In both years, a small-plot belt cone seeder was used to plant the trials at approximately 10 kg ha 1. Every 20th plot in the screening trials was planted to a bromoxynil-susceptible check plot in both years. Emergence counts were conducted approximately 14 d after seeding. Plot stands in all screening trials were vari-

3 CUTHBERT AND MCVETTY OUTCROSSING STUDIES IN OILSEED RAPE 659 able. Consequently, one representative row of each six-row plot was counted in every screening trial. The value obtained for the representative row was multiplied by six to give the plant stand of each screening trial plot. In, the plots were 10 m long and 1.2 m wide at Winnipeg and 8 m long and 1.2 m wide at Carman. In 1998, the plots were 15 m long and 1.2 m wide in the first screening trial at Carman and 10 m long and 1.2 m wide in the second screening trial also at Carman. Plot size was selected in each case to optimize available land area while providing a statistically adequate number of seedlings in each plot. The screening trials consisted of samples from the plot-to-plot trials and the row-to-row trials harvested in. Four hundred and forty plots were planted in an area approximately 0.6 ha. With an average seedling emergence rate of 42%, approximately seedlings were screened for bromoxynil resistance in. The 1998 screening trials consisted of samples from the plot-to-plot trials and the row-to-row trials harvested in. Four hundred and fifty-eight plots were planted in an area approximately 0.7 ha. With an average seedling emergence rate of 45%, approximately seedlings were screened for bromoxynil resistance. The screening trials in and 1998 were sprayed twice with bromoxynil at 750 g a.i. ha 1 (2.7 times the recommended rate of 280 g a.i. ha 1 ), the first spraying approximately 21 d after seeding and the second spraying approximately 5 d later. A second application of bromoxynil was performed to confirm that the plants surviving the first spraying were truly resistant and not the result of a spray miss. After the first spray, the number of bromoxynil resistant plants per plot were counted and recorded. The number of surviving plants per plot were recounted after the second spray. The number of resistant plants confirmed by double bromoxynil spraying were compared to the total number of seedlings per plot to calculate the proportion of resistant individuals (i.e. the outcrossing rate) per plot. Assessing Plant-to-plant Outcrossing Rates Screening trials to determine the outcrossing rates for the plant-to-plant trials were conducted in growth rooms at the University of Manitoba. One screening trial was conducted in the fall of to screen the individual pollen recipient plants grown in Winnipeg in, while two screening trials were necessary in the fall of to screen the individual pollen recipient plants grown in Carman, Portage and Winnipeg in. The screening trials were planted on 20 October, 30 October, and 3 November. A minimum of 150 seeds per pollen recipient plant were planted in 60 cell flats with metro mix. Two seeds per cell were grown to the one-leaf stage, then the number of emerged seedlings was counted just prior to spraying with bromoxynil at 560 g a.i. ha 1 (two times recommended rate) using a cabinet sprayer. The rate of bromoxynil for the indoor screening trial was reduced compared to the field screening trials since seedlings grown indoors under optimum conditions are easier to kill with most herbicides including bromoxynil (pers. comm., Lyle Friesen, University of Manitoba). The flats were sprayed approximately 10 to 14 d after planting. The number of seedlings surviving 2 d after spraying with bromoxynil was counted. The number of bromoxynil-resistant seedlings was compared with the total number of seedlings emerged in the flats to determine the outcrossing rate. Approximately 9970 seedlings in total were screened indoors for the presence of the bromoxynil resistance trait in and. Statistical Analysis Data from the outcrossing trials were subjected to analysis of variance techniques (SAS System for Windows v 6.21, SAS Institute, Inc., Cary, North Carolina, USA). The plotto-plot trials and row-to-row trials used a randomized complete block design at each location in each year. The plot-to-plot trials and row-to-row trials were combined over locations and years and analyzed as a split-split plot design. The plant-to-plant trials used a completely randomized design at each location in each year. The plant-to-plant trials were combined over locations and years and analyzed as a split-split plot design. It was of interest to compare treatment means using the combined plot-to-plot and row-to-row data set. Error variances for the 40-, 80-, 120-cm row spacings and the plot-toplot design were subjected to Bartlett s test for homogeneity of variances (Gomez and Gomez 1984). Since the error variances were heterogeneous and transforming the data set (square root and arc sine square root) did not correct this condition, a conservative procedure to test the significance of the main factors and interactions of the complete data set was followed as suggested by Cochran and Cox (1957). The procedure involved using the mean square error variance and associated degrees of freedom from the data subset with the highest error variance (40-cm row spacing) to test the significance of the main factors and interactions of the complete data set. This mean square error was used in calculating an LSD (0.05) value to separate the treatments. This results in a very conservative estimate of significant differences because this case is the most unfavorable that occurred. The complete data set was then analyzed as a splitsplit-plot over years and sites grown with replicate by year used as the error a term. A paired t-test was performed to determine if the outcrossing rates for each treatment type were significantly different from zero, at the 0.05 level. Outcrossing rates have been measured in this study as the rate of contamination of bromoxynil-susceptible plots, rows and plants by pollen from bromoxynil resistant plots, rows and plants, respectively (i.e. unidirectional outcrossing or pollen mediated gene flow). RESULTS AND DISCUSSION Analysis of variance of the combined plot-to-plot and rowto-row outcrossing data indicated that location and treatments were significant. Location and treatment type accounted for approximately 27 and 53% of the treatment sums of squares for main factors, respectively. The only significant interaction was location by year, which accounted for less than 6% of the treatment sums of squares for main

4 660 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Outcrossing rates in Brassica napus summer rape for plot to plot trials, Carman, Portage and Winnipeg, Manitoba, and seedlings screened Year and location (%) error (Min %) (Max %) (actual no.) (estimated no.) Carman 4.8b Portage 1.9c Winnipeg 6.2a Carman 3.5b Portage 3.7ab Winnipeg 4.7a Carman 4.1b Portage 3.0c Winnipeg 5.4a a a a c s (under each heading) followed by the same letter are not significantly different at the 5% level. factors. Therefore, only the main effects of location, treatment and year are discussed in this paper. Plot-to-plot and Row-to-row Outcrossing Rates The counts of surviving seedlings after the first and second bromoxynil applications were virtually identical, indicating that bromoxynil spray coverage and herbicide activity were satisfactory and predictable. Furthermore, seedlings in the bromoxynil-susceptible check plots were all killed with no seedlings escaping the herbicide application. Plot-to-plot Outcrossing Rates In all cases, the mean plot-to-plot outcrossing rates were significantly different from zero (Table 1). plot-to-plot outcrossing rates were significantly different for all three locations in and for two out of three locations in., Winnipeg had the highest mean outcrossing rate while Portage had the lowest mean outcrossing rate. The difference in outcrossing rates over locations may have been due to differences in pollinator activity, since Winnipeg and Carman had bee hives within 2 km of the trials in both years, while Portage did not have any bee hives in the neighborhood. In contrast, combined over locations, mean outcrossing rates were not significantly different between years. The mean plot-to-plot outcrossing rate combined over locations and years was 4.0% (± 0.23). Of the summer or winter oilseed rape outcrossing studies published to date, only one has investigated plot-to-plot outcrossing. Hühn and Rakow (1979) measured plot-to-plot outcrossing in five low erucic acid cultivars of winter oilseed rape using five cultivars of winter oilseed rape with elevated erucic acid content as a marker. Eighteen plants were selected from the low erucic acid plots and 80 seeds from each plant were analyzed individually for erucic content using paper chromatography. They reported plot-to-plot outcrossing of 2.8 to 7.7% in northern Germany. These results bracket the 4.0% plot-to-plot outcrossing rate observed in the present study. Row-to-row Outcrossing Rates For 40-cm Spacing In all cases, the mean row-to-row outcrossing rates were significantly different from zero (Table 2). row-to-row outcrossing rates ranged from 4.6% (± 0.32) at Portage in to 13.1% (± 1.82) at Winnipeg in, with an overall mean of 9.5% (± 0.62). The Portage location was lower in outcrossing rate than both Carman and Winnipeg in and lower than Winnipeg in. The difference in outcrossing rates over locations may also reflect the differences in pollinator activity., Carman and Winnipeg had similar mean outcrossing rates while Portage had a lower mean outcrossing rate for the 40-cm row-to-row treatment. In contrast, combined over locations, there were no significant differences in outcrossing rates between years. The rows are close enough together in the 40-cm row spacing trial to permit plant-to-plant contact for pollen movement, in addition to pollen movement effected by insect pollinators or wind. For 80-cm Spacing In all cases, the mean row-to-row outcrossing rates were significantly different from zero (Table 3). row-to-row outcrossing rates ranged from 2.2% (± 0.15) at Portage in to 8.2% (± 1.00) at Winnipeg in, with an overall mean of 5.6% (± 0.37). The Portage location was lower in outcrossing rate than both Carman and Winnipeg in and lower than Winnipeg in.

5 CUTHBERT AND MCVETTY OUTCROSSING STUDIES IN OILSEED RAPE 661 Table 2. Outcrossing rates in Brassica napus summer rape for 40-cm spacing row to row trials, Caman, Portage and Winnipeg, Manitoba, and seedlings screened Year and location (%) error (Min %) (Max %) (actual no.) (estimated no.) Carman 10.9a Portage 4.6b Winnipeg 12.2a Carman 9.5ab Portage 7.5b Winnipeg 13.1a Carman 10.4a Portage 5.5b Winnipeg 12.5a a a a, b s (under each heading) followed by the same letter are not significantly different at the 5% level. Table 3. Outcrossing rates in Brassica napus summer rape for 80-cm spacing row to row trials, Carman, Portage, Winnipeg, Manitoba, and seedlings screened Year and location (%) error (Min %) (Max %) (actual no.) (estimated no.) Carman 6.3b Portage 2.2c Winnipeg 7.9a Carman 4.0b Portage 5.2b Winnipeg 8.2a Carman 5.3b Portage 3.6b Winnipeg 8.0a a a a c s (under each heading) followed by the same letter are not significantly different at the 5% level., Carman and Portage had similar mean outcrossing rates, while Winnipeg had a higher mean outcrossing rate for the 80-cm row-to-row treatment. In contrast, combined over locations, there were no significant differences in outcrossing rates between years. As for the 40-cm spacing, the rows in the 80-cm spacing were close enough together to permit plant-to-plant contact for pollen movement, in addition to pollen movement effected by insect pollinators or wind. For 120-cm Spacings In all cases, the mean outcrossing rates were significantly different from zero (Table 4). outcrossing ranged from 2.2% (± 0.15) at Portage in to 6.5% (± 0.52) at Winnipeg in, with an overall mean of 3.9% (± 0.25). The Portage location was lower in outcrossing rate than Winnipeg in and. The cause of the low outcrossing rate at Carman in is unknown.

6 662 CANADIAN JOURNAL OF PLANT SCIENCE Table 4. Outcrossing rates in Brassica napus summer rape for 120-cm spacing row to row trials, Carman, Portage and Winnipeg, Manitoba, and seedlings screened Year and location (%) error (Min %) (Max %) (actual no.) (estimated no.) Portage 2.2b Winnipeg 6.5a Carman 2.4c Portage 3.3b Winnipeg 5.3a Carman 2.4b Portage 2.7b Winnipeg 5.6a a a a, b s (under each heading) followed by the same letter are not significantly different at the 5% level. Table 5. Outcrossing rates in Brassica napus summer rape for plot to plot and row to row trails combined over locations and years Location- seedlings screened Plot type (%) error (Min %) (Max %) years (actual no.) (estimated no.) Plot-to-plot 4.0c Row-to-row 40 cm 9.5a cm 5.6b cm 3.9c Grand total a c s followed by the same letter are not significantly different at the 5% level., Carman and Portage had similar mean outcrossing rates while Winnipeg had a higher mean outcrossing rate for the 120-cm row-to-row treatment. In contrast, combined over locations, there were no significant differences in outcrossing rate between years. The rows are far enough apart in the 120-cm row spacing trial to eliminate plant-to-plant contact, suggesting that pollen movement in this treatment must be effected by insect pollinators or wind. Comparison of Plot-to-plot and Row-to-row Outcrossing Rates Treatment (i.e., plot-to-plot and row-to- row) had a significant effect on the mean outcrossing rates observed for the different treatments studied for trials combined over locations and years (Table 5). outcrossing rates observed in the row-to-row trials were significantly different for all row spacing treatments, with the 40-cm row spacing treatment highest, the 80-cm row spacing treatment intermediate and the 120-cm row spacing treatment lowest. These results indicate that increasing the spacing between nursery rows significantly decreases outcrossing rates. The mean outcrossing rates for the plot-to-plot treatment were significantly lower than the 40- and 80-cm row spacing treatments, but not different from the 120-cm row spacing treatment. Plant-to-plant Outcrossing Rates In all cases, the mean plant-to-plant outcrossing rates were significantly different from zero (Table 6). plant-toplant outcrossing rates ranged from 14.8% (± 1.78) at Portage in to 26.4% (± 3.84) at Winnipeg in, with an overall mean of 21.0% (± 1.73). The Portage location was lower in outcrossing rate than Winnipeg in. The Winnipeg location had high plant-to-plant outcrossing rates in both years, possibly reflecting the enhanced pollinator activity., Winnipeg had a mean plant-toplant outcrossing rate of 25% (± 2.30). As expected, outcrossing rates on an individual plant basis were quite variable, ranging from 5.8% at Portage in to 81.1% at Carman in. The outcrossing rate of 81.1% was verified to determine if it truly was the result of outcrossing because the maximum previously reported plant-to-plant outcrossing rate in oilseed rape is 75%

7 CUTHBERT AND MCVETTY OUTCROSSING STUDIES IN OILSEED RAPE 663 Table 6. Outcrossing rates in Brassica napus summer napus for plant to plant trials, Winnipeg, Manitoba, and Carman, Portage and Winnipeg, Manitoba, Plants screened seedlings screened Year and location (%) error. (Min %) (Max %) (no.) (actual no.) (estimated no.) Winnipeg Carman 19.5ab Portage 14.8b Winnipeg 23.2a Winnipeg a, b s (under each heading) followed by the same letter are not significantly different at the 5% level. (Rakow and Woods 1987). The F 2 progeny (from 20 resistant putative F 1 plants detected in the seed of the 81.1% outcrossed plant), segregated with respect to the resistance trait and confirmed that the estimated 81.1% outcrossing rate for this plant was correct. The majority of oilseed rape outcrossing rate studies published involved plant-to-plant outcrossing, one pollen receptor plant surrounded by several pollen donor plants, similar to the design used in the current plant-to-plant trial. In Sweden, Olsson (1960) studied plant outcrossing rates using petal color as the phenotypic marker. One white (recessive) flowered oilseed rape plant was completely surrounded by yellow (dominant) flowered oilseed rape plants. He reported a mean plant-to-plant outcrossing rate of 34.6% in winter oilseed rape. Persson (1956 as cited by Lewis and Woods 1991) completed a similar outcrossing rate study in winter and spring oilseed rape with petal color as the marker. He observed a mean plant-to-plant outcrossing rate of 28.5% in winter oilseed rape and 36.0% in spring oilseed rape. The results of these studies are somewhat higher than the mean plant-to-plant outcrossing rate of 21.0% obtained in the current plot-to-plant study. The difference in outcrossing rates may be due to the environment studied or pollinator activity and may also be due to the sample size and marker used. Lewis and Woods (1991) indicated the results of the outcrossing studies that used petal color as a marker may be biased because honeybees are sensitive to flower color. Becker et al. (1992) investigated plant-to-plant outcrossing rates in oilseed rape using isozyme polymorphisms in five locations (three sites in Sweden, one site in Denmark, and one site in northern Germany ) to determine if location had an effect on outcrossing rate. At each location, 30 seeds from eight plants were analyzed for polymorphisms. Outcrossing rates, across the locations, ranged from 12 to 47% with a mean outcrossing rate of 34%. The lowest rate of outcrossing (12%) was observed in the most southern location (northern Germany), and the other sites ranged between 32 and 47%. Becker et al. (1992) demonstrated that environment affected plant-to-plant outcrossing rates, which agrees with the current plant-to-plant study because differences in mean outcrossing rates were detected between the Portage and Winnipeg locations in. The mean plant-to-plant outcrossing rate reported by Becker et al. (1992) is higher than the mean outcrossing rate obtained in the current plant-to-plant outcrossing trials. The difference may be due to the different environments or pollinator activity, but may also reflect differences in sample size and the marker used. In Scotland, Gowers (1981) used stem color [purple (dominant) and green (recessive)] differences to measure plant-to-plant outcrossing rates in three cultivars of oilseed rape. Approximately 250 seedlings of each cultivar were evaluated for the presence or absence of purple stem color. Gowers (1981) reported a mean outcrossing rate of 18% based on 750 seedlings screened, a figure that is very close to the mean outcrossing rate of 21% based on 9970 seedlings screened in the plant-to-plant trials reported in this study. On an individual plant basis, Gowers (1981) reported a maximum plant-to-plant outcrossing rate of 38% while the maximum outcrossing rate in the current plant-to-plant trials was 81.1%. The higher rate of plant-to-plant outcrossing observed in the present study may have been due, in part, to excellent synchrony of the pollen recipient and pollen donor near-isogenic populations, in contrast to the different cultivars used by Gowers, which may not have been fully synchronized for flowering. The difference may have also been due to the different environments studied, sample size and marker used. Gowers (1981) conducted his outcrossing experiment at one site in Scotland during one growing season, while the current plant-to-plant outcrossing trials were conducted at four different locations over 2 yr. The outcrossing rates observed in the current plant-toplant trials closely resemble the outcrossing rates reported by Rakow and Woods (1987). Rakow and Woods (1987)

8 664 CANADIAN JOURNAL OF PLANT SCIENCE used elevated erucic acid content in seed oil to measure the outcrossing rate of oilseed rape in Saskatchewan over a 3-yr period. They measured the seed erucic acid content on individual seeds with gas liquid chromatography. In total they screened 3400 seeds from 92 low erucic acid plants. The mean interplant outcrossing rate of 21.8 % reported by Rakow and Woods (1987) closely agrees with the mean interplant outcrossing rate of 21.0% based on the 9970 seedlings screened in the present plant-to-plant outcrossing trials. The maximum outcrossing rate of individual plants was 75% in the study by Rakow and Woods (1987), similar to the 81.1% maximum for outcrossing observed in our plant-to-plant trial. Lewis and Woods (1991) also used erucic acid content to measure plant-to-plant outcrossing in oilseed rape over a 2-yr period in Alberta. An average of 20 seeds from each of 243 plants in 1988 and 24 seeds from each of 35 plants in 1990 were analyzed individually using paper chromatography to detect the presence or absence of erucic acid. They reported a mean interplant outcrossing rate of 55% in 1988 and 45% in 1990, which is much higher than seen in the interplant outcrossing trials in this study (21%). The study by Lewis and Woods (1991) involved two different environments, possible different pollinator activity and a different marker. It is, therefore, not possible to determine which factor(s) account for the observed differences in interplant outcrossing rates between the Lewis and Woods (1991) study and the current study. A significant outcrossing rate was detected and measured for all treatments used in this study. The mean plot-to-plot outcrossing rate was 4.0% while mean row-to-row outcrossing rates were 9.5% for 40-cm row spacing, 5.6% for 80-cm row spacing and 3.9% for 120-row spacing. These outcrossing rates are for simulated plant breeding program treatments. This study, therefore, indicates that the genetic purity of breeding lines harvested at the end of the growing season could be significantly reduced compared to the genetic purity of the seed planted, for typical oilseed rape breeding programs, if pollination control measures are not used. Within a few generations a considerable genetic shift could occur. These results indicate that outcrossing does occur for all treatments used in typical summer oilseed rape plant breeding field layouts and that contamination of breeding lines will occur if pollination control measures are not utilized. A consideration of methods to minimize this contamination is warranted. The use of tents in the field is the best method to eliminate pollen-mediated gene flow between genetically different lines of oilseed rape to maintain the genetic purity of breeding lines. Individual plants can also be bagged to ensure selfpollination. However, this pollination control method is labor intensive and the bagged plants may suffer from disease and insect problems. In the absence of such pollination control measures, nursery rows should be spaced at least 80 cm apart to prevent mechanical crossing and genetically similar breeding lines grouped together in the field. This may help reduce genetic drift for important agronomic and/or seed quality characteristics, but may increase the amount of land required to evaluate breeding lines. An additional way to maintain genetic purity is to use remnant seed from greenhouse or isolated field seed increases for each field season. Genetic purity would be maintained, however, a generation of genetic advance per year may be lost. Doubled-haploid line development techniques can be used in oilseed rape to produce lines completely homozygous at all loci and eliminate up to six segregating generations used in a conventional pedigree selection breeding program. Remnant seed of doubled-haploid lines would maintain genetic purity and a generation of genetic advance would not be lost. In this study, the outcrossing rate of oilseed rape has been measured as the rate of contamination of bromoxynil susceptible plots, rows and plants by pollen from bromoxynil resistant plots, rows and plants, respectively. Most other studies published in the literature have also measured only unidirectional outcrossing. Total (i.e., bidirectional) outcrossing involves pollen mediated gene flow in two directions. The use of two different, dominant, herbicide resistances (e.g., bromoxynil and glyphosate) as markers would allow breeders to accurately assess the total outcrossing rate of oilseed rape. In the absence of a suitable dual dominant herbicide resistance marker system, as a first approximation, doubling the unidirectional outcrossing rate obtained in this study should give an estimate of the total outcrossing rate observed for oilseed rape in western Canada. ACKNOWLEDGMENTS Financial support for this project was provided by Aventis CropScience Canada Co., (formerly Rhône-Poulenc Canada Inc.). The assistance of Lynn Coleman, Patricia Cuthbert, Sandra Fuller, Lyle Friesen, and Robert Smith is also gratefully acknowledged. Becker, H. C., Damgaard, C. and Karlsson, B Environmental variation for outcrossing rate in rapeseed (Brassica napus). Theor. Appl. Genet. 84: Cochran, W. G. and Cox, G. M Analysis of the results of a series of experiments. Pages in Experimental designs. 2nd ed. John Wiley and Sons Inc., London, UK. Eisikowitch, D Some aspects of pollination of oilseed rape (Brassica napus L.). J. Agric. Sci. 96: Gomez, K. A. and Gomez, A. A Test for homogeneity of variance. Pages in Statistical procedures for agricultural research. John Wiley and Sons, London, UK. Gowers, S Self-pollination in swedes (Brassica napus ssp. rapifera) and its implications for cultivar production. 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