The phylogenetic footprinting and association analyses resulted in the identification of polymorphisms and regions that significantly altered FLM expression when present in a homogenous molecular and genomic context. To correlate FLM expression with flowering, we measured flowering time in T2 transgenic lines of eight variants with significantly different FLM abundance (Figure 5A and Figure 5—figure supplement 1A–C). We detected a very strong correlation between FLM-ß transcript levels and flowering time (R2 = 0.94) in plants grown at 15°C. Flowering time responded in a linear manner to FLM-ß levels with 17 to 30 rosette leaves until flowering in the range of relative transcript levels from 0.6 to 2.4 (Figure 5B). Further, the low expression variants PolyA_6xΔ, INT1Δ373bp, and PROΔ225bp flowered as early as the deletion accession Nd-1 suggesting that there must be a critical lower threshold for FLM-ß to be effective (Figure 5A,B). The correlation was much lower for FLM-δ (R2 = 0.70) suggesting that FLM-ß is the major determinant for flowering time in these conditions (Figure 5—figure supplement 1D). Within the range of lines examined, we did not detect a saturating effect at the upper expression level.10.7554/eLife.22114.016Figure 5.FLM-ß expression shows high correlation with flowering time of transgenic plants.(A) Box plot of quantitative flowering time analysis (rosette leaf number) of independent T2 transgenic lines at 15°C and long day photoperiod. Ten plants of three replicate pools were analysed for each construct. The data were corrected for the anticipated 25% of non-transgenic Nd-1 segregants (see Figure 5—figure supplement 1D for the uncorrected analysis). Single values are shown as jittered dots, the colour represents the type of variant as introduced in Figure 4D, the median of the FLMCol-0 reference is indicated as dotted line. Wilcoxon rank test: *p≤0.05; **p≤0.01; ***p≤0.001; n.s., not significant. (B) Correlation (simple linear regression) between FLM-ß and qRT-PCR expression data as presented in Figure 5—figure supplement 1A,B and flowering time analysis as shown in (A). Datapoints of INT6+ variants with contrasting FLM expression and FLMCol-0 are designated. The color code corresponds to (A). The variants PolyA_6xΔ, INT1Δ373bp, and PROΔ225bp, which do not respond in a linear manner are shown as dotted circles. The shaded areas indicate the 95% confidence intervals; p<0.0001. Note, that the flowering time data shown in (A) were corrected by removing values of non-transgenic T2 segregants. An analysis using the uncorrected data is shown in Figure 5—figure supplement 1E,F.DOI: http://dx.doi.org/10.7554/eLife.22114.01610.7554/eLife.22114.017Figure 5—figure supplement 1.FLM-ß expression correlates with flowering time in transgenic lines carrying different FLM alleles.(A) and (B) Results from qRT-PCR analyses of FLM-β (A) and FLM-δ (B) expression in ten day-old seedlings of pools (n = 18–40) of independent T2 transgenic lines. Error bars indicate SD of four replicate pools comprising between four and ten transgenic lines or three Col-0 replicates. (C) Photographs of representative 57 day-old plants expressing FLMCol-0 and PRO2+ and INT6+ variants grown at 15°C in long-day photoperiod. Col-0 and Nd-1, which served as genetic background for the transgenic analysis, are shown in each photograph for comparison. Plants of both pictures are from the same experiment but were photographed separately. (D) Correlation (simple linear regression) between FLM-δ expression data as presented in (B) and flowering time as shown in Figure 5A. Data points of INT6+ variants with contrasting FLM expression and FLMCol-0 are designated. p=0.005. The shaded areas indicate the 95% confidence intervals. (E) Box plot of quantitative flowering time analysis (rosette leaf number) of independent T2 transgenic lines at 15°C and long day photoperiod. Ten plants of three replicate pools were analysed for each construct (uncorrected data). Single values are shown as jittered dots, the colour represents the type of variant as introduced in Figure 4D, the median of the FLMCol-0 reference is indicated as dotted line. The 25% of the plants that show a very early flowering that most likely represent the wild type Nd-1 segregants are indicated with a red circle and these were extracted for correction as presented in Figure 5A. Wilcoxon rank test: *p≤0.05; **p≤0.01; ***p≤0.001; n.s., not significant. (F) Correlation (simple linear regression) between FLM-ß and FLM-δ. qRT-PCR expression data as presented in (A) and (B) and flowering time analysis as shown in (D). FLM-ß, p<0.0001; FLM-δ, p=0.002. The shaded areas indicate the 95% confidence intervals.DOI: http://dx.doi.org/10.7554/eLife.22114.017