Botany
A sexual hybrid and autopolyploids detected in seed from crosses between Neslia paniculata and Camelina sativa (Brassicaceae)
Journal: Botany
Manuscript ID cjb-2019-0202.R2
Manuscript Type: Note
Date Submitted by the 03-Mar-2020 Author:
Complete List of Authors: Martin, Sara; Agriculture and Agri-Food Canada, Ottawa Research and Development Centre LaFlamme, Michelle; Agriculture and Agri-Food Canada, Ottawa Research and DevelopmentDraft Centre James, Tracey; Agriculture and Agri-Food Canada, Ottawa Research and Development Centre Sauder, Connie; Agriculture and Agri-Food Canada, Ottawa Research and Development Centre
Brassicaceae, hybridization, autopolyploidization, neopolyploidy, gene Keyword: flow
Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :
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1
2
3 A sexual hybrid and autopolyploids detected in seed from crosses between Neslia paniculata and 4 Camelina sativa (Brassicaceae)
5 Sara L. Martin1, Michelle LaFlamme1, Tracey James1, Connie A. Sauder1
6
7 1 Agriculture and Agri-Food Canada, Ottawa Research and Development Centre, 960 Carling Ave., 8 Ottawa, Ontario, K1A 0C6
9 Corresponding Author: 10 Sara L. Martin 11 960 Carling Ave. Ottawa, Ontario, K1A 0C6 12 613-715-5406 13 [email protected] 14
15 Emails of co-authors: 16 [email protected] Draft 17 [email protected] 18 [email protected]
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19 Abstract
20 It is important to understand the probability of hybridization and potential for introgression of
21 transgenic crop alleles into wild populations as part of pre-release risk assessment. Here we completed
22 bidirectional crosses between the emerging crop, camelina (Camelina sativa) and its weedy relative ball
23 mustard (Neslia paniculata). Ball mustard is a self-compatible annual that produces hard ball-like seeds
24 similar to canola or mustard seed in size and shape. A total of 1,593 crosses were completed and
25 collected with camelina as the maternal parent, while 3,253 crosses were successfully collected in the
26 reverse direction. Putatively hybrid seedlings were screened with flow cytometry and species-specific
27 nuclear ribosomal internal transcribed spacer (ITS) markers. Three plants had DNA contents close to
28 expectations for hybrids, but only one of these, formed on camelina, had the expected ITS markers. This
29 hybrid exhibited low fertility and neither selfDraft pollination nor backcrossing produced viable progeny. The
30 other two plants, formed on ball mustard, had high pollen and seed fertility and were identified as ball
31 mustard neoautotetraploids. Therefore, the hybridization rate between camelina and ball mustard is
32 relatively low at 1 in 20,000 ovules pollinated when camelina is the maternal parent. However,
33 autotetraploids may form frequently in ball mustard and tetraploid populations may exist in nature.
34 Key words: Brassicaceae, hybridization, autopolyploidization, neopolyploids, weeds, gene flow
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35 Introduction
36 Within Canada, numerous plant species could receive gene flow from crops with novel traits. As
37 part of a pre-release risk assessment, it is important to understand the probability of this hybridization
38 and the potential for introgression of crop alleles into wild populations (Anderson 1949; Ellstrand and
39 Hoffman 1990; Dale et al. 1993; Warwick et al. 1999; Snow 2002; Ellstrand 2003; Messeguer 2003;
40 Devos et al. 2009). The short term fitness effects of hybridization and gene flow from crops to the wild
41 population and the long term evolutionary consequences of this introgression will depend on the
42 characteristics of the novel trait (Mallet et al. 2016). However, fundamentally, whether or not a trait can
43 be incorporated into another species’ genome, depends on the strength of reproductive isolation
44 between the lineages. This strength has proven to be highly variable across taxa with hybridity making a
45 substantial contribution to the evolutionaryDraft histories of many plant species (Mallet 2007; Abbott et al.
46 2013).
47 Camelina (Camelina sativa (L.) Crantz; n = 20 (6+7+7) is a hexaploid crop with strong potential to
48 produce high value products such as biofuel and a sustainable source of for omega-3 fatty acids for
49 human consumption (Small 2013; Berti et al. 2016). Several close relatives of camelina, including other
50 species of Camelina, Shepherd’s purse (Capsella bursa-pastoris (L.) Medik.), three species of Arabidopsis
51 (A. arenicola (Richardson ex Hooker) Al-Shehbaz, Elvin, D. F. Murray & Warwick, A. lyrata (L.) O’kane
52 &Al-Shebaz, and A. thaliana (L.) Heynhold), and ball mustard (Neslia paniculata (L.) Desv.) (Al-Shehbaz
53 and co-workers 2010; Al-Shehbaz 2012), have become naturalized in Canada and are weeds of marginal
54 and agricultural lands. Ball mustard is a self-compatible, annual or winter annual plant that, like
55 camelina, is native to central Eurasia and was first introduced into in Manitoba, Canada in the 1800s
56 (Francis and Warwick 2003). The species is generally considered diploid (n = 7) (Mulligan 1957; Al-
57 Shehbaz and co-workers 2010).
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58 Historically, within Canada, ball mustard has been most abundant on the Canadian Prairie
59 Provinces, with occurrences recorded across the country (Francis and Warwick 2003). Ball mustard has
60 small hard ball-like seeds with strong dormancy that are able to persist in the seed bank and, because of
61 similarity in shape and size of their seeds, can be a problematic contaminant of canola (Brassica napus
62 L.) or mustard (Sinapis alba L.). Abundance of ball mustard appears to have declined from being
63 common in the 1880s (Francis and Warwick 2003) to low abundance in the Canadian Prairie Provinces in
64 the 1990s (Leeson et al. 2005). This may be a result of good control of the species with herbicides.
65 However, an Alberta population resistant to acetolactate synthase inhibitors was found in 1998 (Heap
66 2019). The genome has been sequenced (Slotte et al. 2013) and the species has received attention as a
67 system for understanding karyotype evolution in the Brassicaceae (Lysak et al. 2006; Mandáková and 68 Lysak 2008). Ball mustard is indeterminate Draftand flowers from May to September, which would overlap 69 with the flowering period of camelina. Hybridization has been reported between the two subspecies of
70 ball mustard, Neslia paniculata subsp. paniculata and the apparently hexaploid (n = 21) Neslia
71 paniculata subsp. thracica (Velen.) Bornm. (Francis and Warwick 2003; Rice et al. 2015), but we are
72 unaware of any work examining the species’ potential to make wide hybridizations. Here we performed
73 bidirectional crosses between camelina and ball mustard to establish their baseline inter-fertility.
74 Materials and Methods
75 Seed Sources
76 Five accession of camelina including both cultivated material and material collected from feral
77 populations in Canada and four accessions of ball mustard (Neslia paniculata subsp. paniculata) received
78 from botanical gardens and from material collected in Canada were used (Table 1). Seeds of ball
79 mustard had the seed coats removed prior to planting, while camelina seeds were planted directly into
80 soil, into 10 cm pots filled with a 1:2:1 mixture of soil, peat and sand. These were placed in the green
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81 house with a 16 h photoperiod with supplemental lights programed to come on when light levels are
82 below 400 W/m2 and temperatures of 18 °C at night and 20 °C during the day.
83 Controlled Crosses
84 Three treatments were applied to each plant. Buds were emasculated and after a day were 1)
85 left un-pollinated as negative controls, 2) pollinated with self-pollen for a positive control or 3)
86 pollinated with the pollen of the other species as the crossing treatment. Further, un-manipulated
87 flowers from each plant were selected as a second positive control. As camelina has approximately 8-20
88 ovules per flower, we expected that 1000 pollinations with camelina as the maternal parent would
89 result in approximately 12,000 ovules challenged by ball mustard pollen and the power to detect 90 hybridization at a rate of 0.025% with 95% Draftconfidence (Jhala et al. 2011). In contrast, while the 91 gynoecium of ball mustard initially contains 4-6 ovules, only one, occasionally two, and more rarely,
92 three, develop into seeds. This limits the expected number of ovules/per pollination to one. As a result,
93 a thousand pollinations only results in a thousand trials for hybridity and 2,995 pollinations are required
94 for detecting a hybridization rate of 0.1% with 95% confidence (Jhala et al. 2011). Any seed that formed
95 on flowers that received foreign pollen were treated like the seeds of their maternal parent and grown
96 under the same conditions.
97 Hybrid Screening
98 Flow cytometry was used to screen putative hybrids between ball mustard and camelina based
99 on their DNA content following protocols described previously (Martin et al. 2015, 2017). Briefly,
100 samples were fresh leaf material from rosettes that were collected in the greenhouse and placed on ice,
101 chopped with a new, sharp razor blade in Galbraith buffer (0.75 mL), and then kept in the dark while
102 they stained for 30– 40 min with propidium iodide. Samples were run with radish (Raphanus sativus L.
103 ‘Saxa’ (1.11pg/2C)) as an external standard for initial screening, however camelina was used as an
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104 internal standard for precise DNA estimates with three technical replicates completed over three
105 different days (Doležel et al. 1992). Samples were run on at low speed on a Gallios flow cytometer
106 (Beckman Coulter, Ontario, Canada). The DNA content of samples was estimated using a fluorescence
107 585/42 nm detector and all characteristics of the fluorescence area peaks including the mean,
108 coefficient of variation and nuclei counts were determined using the R package flowPloidy (Smith et al.
109 2018). Camelina has a 2C DNA content of 1.59 pg (Martin et al. 2017) and we determined that ball
110 mustard has a 2C DNA content of 0.43 ± 0.01 pg (n = 10). As a result, homoploid hybrids between the
111 species were expected to have 2C DNA contents of approximately 1.01 pg.
112 Additionally, species specific PCR markers from the nuclear ribosomal internal transcribed
113 spacer (ITS) were developed to confirm hybridity using selective forward primers (Nes ITS 112F (5’-
114 CGGATCCGTGGTTTCGCGTGC -3’) and Cam DraftITS 112F (5’- TGCCGTTTCCGTGGTTTCGCGTATC -3’) ) and a
115 non-selective reverse primer P4 Cam (5’- TTTTCCTCCGCTTATTGATAT G -3’). Amplification reactions
116 contained 1.0 µl of genomic DNA, 0.2uM Forward and Reverse primers, PCR buffer at 1X, 0.2uM dNTPS,
117 and 0.01 unit of Hotstart Taq polymerase (Qiagen, Germantown, MD, USA). The PCR protocol consisted
118 of 15 min at 95 °C followed by 30 cycles of 95 °C for 30 s, annealing (69 °C Nes ITS 112F and Cam ITS
119 112F 67°C) for 30 sec, 72 °C for 30 sec, followed by a 10 min final elongation at 72 °C. Reaction
120 products were separated on a 1.0 % agarose gel and stained with Gel Red (Biotium Inc., Fremont, CA).
121 Hybrids would therefore be expected to show the approximate 400 bp marker from both parents.
122 Characterization of the hybrid
123 Pollen fertility and backcrossing success were estimated for the hybrid produced in this
124 experiment. The pollen fertility was determined by staining with a 0.5% solution of acetocarmine and
125 scoring approximately 1,000 pollen grains as viable or non-viable for three flowers. Crosses were
126 conducted between the hybrid and both camelina (161 with camelina as maternal parent and 472 as the
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127 paternal parent) and ball mustard (149 with ball mustard as maternal parent and 588 with as the
128 paternal parent). All seed produced in these crosses were tested for germination and surviving seedlings
129 were tested for evidence of hybridity. In addition, 1,831 unmanipulated pods that self-pollinated
130 naturally were collected from the hybrid and cleaned. We attempted to germinate all seeds produced.
131 Characterization of seed production and viability
132 In addition to assessing pollen fertility, as completed for the hybrid, seed production and
133 viability were characterized for the autopolyploids, their diploid siblings or parents and triploids
134 produced through cross pollination of the two cytotypes. Seed produced were cleaned using a 1.7mm
135 sieve and both total seed production and three groups of 100 seeds were weighed. Thirty 136 (unmanipulated - naturally self-pollinated) Draftseeds from each individual were sown into pots and 137 monitored for germination for 3 weeks.
138 Statistical Analyses
139 All statistical analyses were conducted in R (v. 3.4.4, "Someone to Lean On" (R Core Team 2017)).
140 Kruskal-Wallis tests were used to compare seed set in treatments with camelina as the maternal parent
141 and pollen production, as the data were non-normal. The seed counts from ball mustard as the maternal
142 parent were similar to a Poisson distribution and were compared using an exact ratio test from the
143 package rateratio.test (Fay 2010). Pollen production and seed weight for diploids and tetraploid ball
144 mustard were compared with one way tests with correction for unequal variances (oneway.test).
145 Residuals from ANOVAs (aov) for seed production, weight and viability for diploid, triploid and tetraploid
146 comparisons did not deviate strongly enough from expectations to reject the validity of the tests and
147 were followed by Tukey’s test. Additional packages used for graphical and plotting functions were plotrix
148 (Lemon 2006) and pgirmess (Giraudoux 2013).
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149 Results
150 Power to Detect Hybridization
151 In total 1,593 crosses were completed and successfully collected between camelina and ball
152 mustard with camelina as the maternal parent, while 3,253 crosses were successfully collected in the
153 reverse direction. Seed set on the positive controls that were emasculated and the self-pollinated plants
154 suggest that, on average, 12.8 ovules were challenged with pollen per camelina flower for an estimated
155 total of 20,264 ovules, giving the power to detect hybridization at a rate of 0.015% with 95%
156 confidence. For ball mustard, on average 0.95 seeds were formed with the self-pollination treatment
157 indicating 3095 ovules were challenged giving the power to detect hybridization rates of 0.098% with 158 95% confidence (Jhala et al. 2011). Draft 159 One Hybrid and Two Autopolyploids Detected Among Seed from Crosses
160 A total of 373 seeds formed on camelina pollinated by ball mustard, while 233 seeds formed on
161 ball mustard flowers that received camelina pollen (Table 2). All of these seeds were sown and those
162 that germinated and persisted to the rosette stage, 354 (95%) from camelina as the maternal parent and
163 180 (77%) from ball mustard as the maternal parent, were tested for genome size using flow cytometry.
164 Three individuals, with nearly intermediate DNA contents were detected, one from a camelina maternal
165 parent (1.00 ± 0.01pg) (CS-05 X NP-03) and two from ball mustard maternal parents NP-03 and NP-04
166 (with 2C values of 0.85 ± 0.07 pg and 0.97 ± 0.01 pg respectively). As these values approximately
167 matched the expectation that the 2C DNA contents of a hybrid would be 1.01 pg, these three individuals
168 were investigated further.
169 The individual from the camelina maternal parent had the ITS marker from ball mustard, in
170 addition to the ITS marker from its maternal parent, and had low pollen fertility (1.08%). This homoploid
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171 hybrid produced: no seed from 588 pollinations with ball mustard pollen; two seeds from 472 flowers
172 pollinated with camelina pollen; and three seeds from 1,831 unmanipulated flowers. However, none of
173 these seeds germinated. Five seeds were produced by the 149 flowers of ball mustard pollinated with
174 the hybrid’s pollen and 103 seeds were produced by the 161 flowers of camelina pollinated with the
175 hybrid’s pollen. However, none of these seeds produced seedlings with DNA contents differing from the
176 expected parental values or showed ITS markers expected for hybrids. This indicates a relatively low
177 hybridization rate of 1 in 20,000 ovules pollinated when camelina is the maternal parent and that near
178 complete sterility can be expected for homoploid hybrids.
179 The two individuals from ball mustard maternal parents with intermediate 2C DNA values had
180 the ITS marker from ball mustard, but did not have the camelina ITS marker (Supplementary Figure 1).
181 Further, did they show any morphological indicationsDraft of hybridity, but rather were nearly
182 indistinguishable from their diploid siblings that had the expected 2C DNA content of 0.43 pg. These
183 individuals also showed similar pollen viability (77.7% and 84.5%) when compared to diploids, which had
184 an average pollen viability of 89.8% (n = 23). Indeed when these two individuals were grouped with 15
185 of their tetraploid progeny there was no difference in pollen viability (F1,34.6 = 0.48, p = 0.4943). Similarly,
186 the total seed weight produced by these two individuals (14.7 and 7.1 g) was not greatly reduced
187 compared to the average total seed weight (9.4 ± 0.7g ) produced by diploids (n = 28). Indeed, when
188 grouped with their progeny the total seed weight produced by these lines were significantly greater
189 than that produced by the diploids (9.4g vs 12.9 g; F1,49.9 = 11.3, p = 0.002). We therefore concluded that
190 these individuals most likely represent autopolyploidization events, rather than hybrids between the
191 species, as ball mustard autopolyploids would be expected to have 2C DNA contents of approximately
192 0.86 pg. The rate of autopolyploidization in ovules challenged with foreign pollen was approximately
193 1:1,500 (2 for 3095 ovules challenged). However, whether autopolyploidization was induced by the
194 presence of foreign pollen, the manipulation of ball mustards’ flowers, or whether it is the result of a
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195 high rate of unreduced gamete production in this species remains to be determined. As a result, we
196 conclude that no hybrids were detected with ball mustard as the maternal parent. If hybridization
197 occurs in this direction, we estimate that it occurs at a rate lower than one in 1,000 ovules challenged.
198 Given the apparently high rate of autopolyploidization in ball mustard, we completed a further
199 1,168 crosses with camelina as the maternal parent using the first generation autopolyploids or their
200 progeny (which were the result of self-pollination of the first generation plants). Putative hybrids (n =
201 243) were then screened with flow cytometry as described above, but no hybrids were detected. In this
202 case our number of pollinations should give the power to detect a hybridization rate of 0.018% with 95%
203 confidence (given an estimated 17,129 ovules tested). This indicates that autopolyploidization does not
204 result in a substantial increase in hybridization rate with camelina as the maternal parent and that
205 hybridization, if it occurs in this direction, wouldDraft occur at less than one in 5,000 ovules challenged.
206 We also completed approximately 100 inter-ploidy pollinations between diploid (150 flowers ♀)
207 and tetraploid (110 flowers ♀) ball mustard to establish whether or not the cytotypes could produce
208 offspring. About a third of these crosses, 32 and 27 for diploid and tetraploid maternal parents,
209 respectively, produced viable triploid seedlings with a 2C DNA content of approximately 0.65 pg. Seed
210 production was evaluated for 25 of these triploid individuals for comparison with diploid (n = 28) and
211 tetraploid (n = 24) ball mustard, while pollen fertility was evaluated for 17 triploids, 30 diploids and 25
212 tetraploids.
213 Fertility and viability of diploid, triploid and tetraploid ball mustard
214 Pollen viability was not significantly different for diploid and tetraploid Neslia, but was
215 significantly lower in triploid Neslia averaging 50.3% according to a Kruskal-Wallis test (Χ2 = 42.5, p <
216 0.001) (Figure 1a). The total weight of seed produced by diploid and triploid ball mustard accessions was
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217 significantly lower than that produced by tetraploids according to an ANOVA (F2,74 = 83.1, p < 0.001).
218 However, the average weight of 100 seeds was significantly different for all three cytotypes with
219 triploids having the lowest weight and tetraploids the highest (F2,74 = 15.2, p < 0.001). When the number
220 of seeds produced by each plant was estimated from these data, the cytotypes showed no significant
221 differences (F2,74 = 1.5, p = 0.224) indicating that tetraploids produced heavier seeds in the same
222 numbers as the diploids (Figure 1b, c). However, the viability of triploid seed was approximately 4%,
223 significantly lower than the fertility of diploids and tetraploids (F2,73 = 108.1, p < 0.001; Figure 1d).
224 Discussion
225 The hybridization rate found between camelina and ball mustard here is relative low, at 1 in 226 20,000 ovules challenged or 0.005%. This isDraft similar to the overall rate of 0.007% inter-compatibility 227 found between Brassicaceae species that form sexual hybrids (FitzJohn et al. 2007). It is lower than
228 hybridization rates found between camelina and shepherd’s purse (Capsella bursa-pastoris (L.) Medik)
229 when camelina is that maternal parent of 1.5 hybrids for 10,000 ovules challenged (Martin et al. 2015).
230 While it is possible that hybridization with ball mustard as the maternal parent will occur at rates below
231 our power to detect in this work, which was limited, given the ploidy difference between the diploid ball
232 mustard and hexaploid camelina (Kagale et al. 2014), this rate may be much lower than that observed
233 with camelina as the maternal parent (Bing et al. 1995; Ramsey and Schemske 1998; FitzJohn et al. 2007;
234 Vallejo-Marin et al. 2016). Additionally, autotetraploidization of ball mustard did not result in a dramatic
235 change in the hybridization rate when camelina was the maternal parent and, if it occurred, likely was
236 fewer than one in 5,000 ovules challenged. Considering the low hybridization rate, the primarily
237 autogamous nature of these two species, the poor fertility of the hybrid produced, and the current
238 distribution of ball mustard in Canada, while hybridization may occur between these species in nature, it
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239 appears to be a lower risk than other species pairings likely to occur in Canada such as camelina with
240 shepherd’s purse.
241 Given our observations of the autopolyploids produced here, it seems possible that ball mustard
242 occurs in diploid, autotetraploid or mixed ploidy populations in nature. Indeed, chromosome count data
243 from material from Canada and Europe have indicated both diploid and tetraploid counts (Mulligan
244 1957; Rice et al. 2015) suggesting that tetraploids do indeed occur in natural populations.
245 Autopolyploidization events have been estimated to occur at a rate of 2.16 X 10-5 though self-pollination
246 (Ramsey and Schemske 1998) and unreduced gamete production has been found to be relatively high in
247 many Brassicaceae species (Kreiner et al. 2017). Given that we detected two autotetraploids in 233
248 seeds screened (or in 3095 ovules pollinated), it appears ball mustard has a high rate of autopolyploid
249 formation. The relatively high pollen and seedDraft fertility of the autopolyploids compared to their diploid
250 progenitors may indicate that the fitness of neoautotetraploids will be high when they arise in nature,
251 increasing the probability of establishment after formation. In addition, crosses between diploid and
252 tetraploid ball mustard seem to readily produce triploids with low, but not negligible, fertility levels. As a
253 result, ball mustard may provide a fruitful and tractable system to understand the initial stages of
254 autopolyploid formation, establishment, and the role of the triploid bridge in facilitating establishment
255 of polyploid populations (Ramsey and Schemske 1998; Husband 2004). Further work is needed to
256 determine if and where autotetraploid populations occur in nature, to investigate their fitness relative
257 to their parents in natural environments, and determine if the autopolyploidization rate seen here was
258 influenced by foreign pollen, manipulation of the flowers, or the limitation of conspecific pollen.
259 Acknowledgements
260 We thank greenhouse staff at ORDC for help raising and caring for plants. We also thank the Meise
261 Botanical Garden in Belgium, the Botanischer Garten at Ulm University in Germany, Dr. Hugh Beckie, Dr.
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262 Linda Hall and for providing Neslia paniculata accessions for this research. This research was funded by
263 AAFC grant “Systematics of Weeds, Crops and Crop Wild Relatives” (J-002275).
Draft
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352 Table 1. Seed sources and origins for material used in this experiment.
Species Code Origin Camelina CS-01 Mortlach, Saskatchewan CS-02 Estevan, Saskatchewan CS-03 North Central Regional Plant Introduction Station Accession Krasnodar Area, Former Soviet Union ID : PI258366 CS-04 Hortus botanicus, Academia scientiarum, Salaspils, Lativa, URSS Accession ID : 3418 CS-05 “Calena” Lethbridge, Alberta, Mercer Seeds Ball mustard NP-01 Saskatchewan, Dr. Hugh Beckie NP-02 Belgium, Meise Botanical Garden NP-03 Germany, Botanischer Garten at Ulm University NP-04 Alberta, Dr. Linda Hall 353
354 Table 2. Seed production in reciprocal crosses and control treatments of camelina and diploid ball 355 mustard.
Treatment Camelina as Maternal Parent Ball Mustard as Maternal Parent Silicles SeedDraft Seed/Silicle Balls Seed Seed/Ball Collected Count Collected Count Emasculation Only 167 52 0.31 258 22 0.09 Emasculation and 170 2168 12.75 351 335 0.95 Self-Pollination Emasculation and 1589 373 0.23 3233 231 0.07 Cross-Pollination Unmanipulated 223 2686 12.04 254 268 1.06 356
357
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358 Figure 1. Mean and standard error for a) percent pollen viability for diploid (n = 30), triploid (n = 25) and 359 tetraploid (n = 17) ball mustard plants, b) average 100 seed weight (mg) (diploid (n = 28), triploid (n = 25) 360 and tetraploid (n = 24), c) total estimated seed count (diploid (n = 28), triploid (n = 25) and tetraploid (n 361 = 24)), and d) seed viability for 30 seeds planted in pots and monitored for one month for naturally self- 362 pollinated seeds from diploid (n = 28), triploid (n = 25) and tetraploid (n = 24). Letters indicate 363 significance as determined by a Tukey's Honest Significant Difference test following ANOVAs (p < 0.05).
Draft
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Draft
Mean and standard error for a) percent pollen viability for diploid (n=30), triploid (n=25) and tetraploid (n=17) ball mustard plants, b) average 100 seed weight (mg) (diploid (n=28), triploid (n=25) and tetraploid (n=24), c) total estimated seed count (diploid (n=28), triploid (n=25) and tetraploid (n=24)), and d) seed viability for 30 seeds planted in pots and monitored for one month for naturally self-pollinated seeds from diploid (n=28), triploid (n=25) and tetraploid (n=24). Letters indicate significance as determined by a Tukey's Honest Significant Difference test following ANOVAs (p < 0.05).
181x236mm (300 x 300 DPI)
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