Following on from the positive identification of both BoFLC2 and boflc2 alleles in cauliflower lines, four different parent lines with suitable physiological and commercial qualities were selected for further examination. A subset of plants from the late-flowering parents A and C were identified as possessing homozygous functional BoFLC2 (alternatively referred to as +/+ for the remainder of this chapter) and a subset of plants from early- flowering parents B and D were found to possess homozygous boflc2 (alternatively referred to as -/- hereafter). These results are shown in Figure 6. Subsequent genotyping of the entire population of field-grown parental lines identified one and three heterozygous (+/-) plants in the S2 and S3 generations of the A parent line, respectively. One heterozygous plant was
observed out of the total 30 plants in the S6 parent line C. Heterozygous parent lines were
excluded from all subsequent statistical analyses. All plants in the B and D parent lines were homozygous for the mutant boflc2, and all CD F1 plants were heterozygous for this allele.
Genotyping of the AB F2 populations revealed a segregation of 102:175:97 (homozygous
functional:het:homozygous mutated) for the BoFLC2 gene. This conforms to the expected theoretical 1:2:1 distribution ratio of 93.5:187:93.5 (df = 2; 2crit with at 0.01 = 9.21; 2obs =
1.67). In the CD F2 population, a segregation of 120:186:71 (homozygous
functional:het:homozygous mutated) was observed, representing a significant departure from the theoretical expected distribution ratio of 94.25:188.5:94.25 (df = 2; 2crit with at 0.01 =
9.21; 2obs = 12.80).
Figure 9 shows that the average length of time until curds became visible was significantly (P<0.0001) different between the three BoFLC2 genotypes in the segregating AB F2
population (Kruskal-Wallis one-way ANOVA). The average length of time from transplanting until curd visibility in +/+ F2 plants was 88.2 days, compared to 84.6 days in +/-
and 80.0 days in -/- plants. This effect was more extreme in the parent lines, with the S2
parent line A curding after 122.3 days and the S6 parent line B curding after just 78.0 days.
These figures are significantly different from those for the corresponding genotypes in the segregating F2 population (P<0.001). Also, in the S3 A parent line, curds appeared
significantly faster (116.5 days) than in the A parent line that was only inbred for two generations. Approximately 84% of -/- plants in the F2 population had already formed curds
Chapter III The Contribution of BoFLC2 to Flowering Time in Cauliflower
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plants had formed curds by the first week, followed by approximately 48% of +/+ plants. Several +/+ and +/- plants in the F2 population and a number of plants in both A parent lines
had not produced visible curds at the time of the final recording.
Fig. 9. Average number of days from transplanting until curds were visible in three BoFLC2 genotypes in late-flowering parent A, early-flowering parent B, an S3 population of parent A, and a segregating F2 population. +/+ denotes plants with homozygous BoFLC2, -/- denotes plants with homozygous
boflc2, and +/- denotes heterozygotes. Bars represent the standard error of the mean.
Figure 10 shows that in the second (CD) F2 population, the average length of time until curds
became visible was also significantly different between the three BoFLC2 genotypes. The average length of time from transplanting until curd visibility in +/+ F2 plants and parent line
C was 94.5 and 90.7 days, respectively; in -/- plants, the duration was an average of 82.1 days and 81.2 days for parents. F2 +/- curds became visible after an intermediate period of 88.0
days; however, in the heterozygote F1 generation, curds became visible after just 80.1 days –
a similar period of time to the D parent. Significant (P<0.0001) differences in time until curd visibility were observed between all three genotypes in the F2 population (Kruskal-Wallis
one-way ANOVA). Significant (P<0.0001) differences were also observed between the C/C parent plant and the other two genotypes, but no significant difference was found between the C/D genotype and the D/D genotype. Around 64% of the -/- plants had already developed a visible curd 78 days after planting, in comparison to 29% of the +/- plants and only 12% of the plants with the +/+ genotype. Only one plant (a heterozygote) had not initiated a visible curd by the final week of measurement.
70 80 90 100 110 120 Parents/F3 F2 A ve rage N o . D ay s U nti l C urd A ppe ara nc e Segregating F2 Progeny
Par. A (S2) Par. A (S3) Par. B
Chapter III The Contribution of BoFLC2 to Flowering Time in Cauliflower
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Fig. 10. Average number of days from transplanting until curds were visible in three BoFLC2 genotypes in late-flowering parent C, early-flowering parent D, F1 plants and a segregating F2 population. +/+ denotes plants with homozygous BoFLC2, -/- denotes plants with homozygous boflc2, and +/- denotes heterozygotes. Bars represent the standard error of the mean.
Comparison of developmental stage between genotypes in the CD population was conducted at the fixed point of 117 days after transplanting. Figure 11 shows that at this point, F2 plants
with homozygous BoFLC2 had an average developmental stage of 2.6, corresponding to a „loose‟ curd just prior to peduncle elongation (see Figure 3 for curd development key). Heterozygous plants had an average stage of 3.6, which corresponds to a curd with elongating peduncles which has not quite formed distinct flower buds. An average developmental stage of 5.0 was observed in plants with homozygous boflc2 corresponding to a flowering plant with less than 12 flowers fully open. The developmental stages of parent lines and plants from the F1 population were similar to their corresponding genotypes in the F2 population
(average stages of 2.4, 4.3 and 4.8 in parent C, F1 and parent D, respectively). Significant
(P<0.0001) differences in development between the three genotypes were observed in both the F2 population and the parent lines/F1 plants (Kruskal-Wallis one-way ANOVA).
Although variance within genotypes was found to be homogenous (Bartlett‟s test), a Shapiro- Wilk test failed to support the null hypothesis that the data were normally distributed.
70 75 80 85 90 95 100 Parents/F1 F2 A ve rage N o . D ay s U nti l C urd A ppe ara nc e Segregating F2 Progeny Parent C F1 Parent D +/+ +/- -/- +/+ +/- -/-
Chapter III The Contribution of BoFLC2 to Flowering Time in Cauliflower
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Fig. 11. Average developmental stage of curds in three BoFLC2 genotypes in late-flowering
parent C, early-flowering parent D, F1 plants and a F2 segregating population 117 days after transplanting. +/+ denotes plants with homozygous BoFLC2, -/- denotes plants with homozygous boflc2, and +/- denotes heterozygotes. Bars represent the standard error of the mean. Developmental stage figures are based on Figure 3.
Curd size was recorded throughout the observation period in CD F2 plants. In the first week
of measurement (78 days after planting), the average curd size of -/- plants was 11.1 cm, greater than that of the +/- plants (5.2 cm), which in turn was greater than the average curd size of +/+ plants (2.0 cm; see Figure 12). In the -/- genotype, curd size increased steadily before reaching a plateau. The other two genotypes appear as though they would also follow this general trend, but recording ceased before a clear plateau was reached. Despite significant differences (P<0.0001) in average maximum observed curd size (-/- = 26.7 cm; +/- = 25.0 cm; +/+ = 22.1cm) it appears that all three genotypes were tracking to reach a roughly similar maximum size, and by fitting quadratic trend lines to the data, similar maximum average curd diameters are predicted for each genotype (24.9 cm, 23.1 cm and 25.9 cm for -/- , +/-, +/+, respectively). However, while the homozygous mutant boflc2 lines would be predicted to reach this maximum size after 108 days, the heterozygotes would require a further 6 days to reach this predicted size, and the homozygous functional lines would require yet another 26 days. Using a separate slopes model and comparing the slope between average Y values at the first two time points in the -/- line and the average Y values at the first three points in the +/- and +/+ lines, the slopes (indicating rate of curd growth) in the three genotypes were significantly different (P=0.0427), with the rate of curd growth slower in lines with more functional copies of BoFLC2.
0 1 2 3 4 5 6 Parents/F1 F2 D ev . Sta ge 117 da ys af te r tra ns pl an ti ng Segregating F2 Progeny Parent C F1 Parent D +/+ +/- -/- +/+ +/- -/-
Chapter III The Contribution of BoFLC2 to Flowering Time in Cauliflower
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Fig. 12. Average curd size in the CD segregating F2 population. +/+ denotes plants homozygous for BoFLC2, -/- denotes plants homozygous for boflc2, and +/- denotes heterozygotes. Polynomial trend lines are displayed, along with slopes of linear regression lines for early time points (first two for -/- and first three for +/- and +/+). Bars represent the standard error of the mean.