Introns 2–6 were required for temperature-sensitive FLM expression (Figure 1). When we statistically tested the interaction between genotype and temperature with a multiple linear model, we found that temperature-sensitive FLM-ß regulation was significantly reduced in INT6+CAA between 15°C and 23°C and when compared to the FLMCol-0 control variant (p=0.012) (Figures 4E and 6A,B). In line with the prediction, flowering of this variant was indeed less sensitive to temperature changes when tested at 15°C and 23°C in homozygous T3 progeny plants and compared to the FLMCol-0 reference (Figure 6C,D). Thus, temperature-independent FLM-ß expression changes correlate with temperature-insensitive flowering in the selected temperature range.10.7554/eLife.22114.018Figure 6.The INT6+CAA polymorphism reduces temperature-sensitivity of FLM expression and flowering.(A) qRT-PCR analysis and (B) expression ratios of FLM-β of FLMCol-0 (INT6+AAT) and INT6+CAA grown in 15°C and 23°C. Statistical tests of temperature-sensitivity are described in the text. (C) Means and SD of quantitative flowering time analysis (rosette leaf number). n = 5 (15°C) and 8 (23°C) replicates from five independent homozygous T3 transgenic lines grown at 15°C and 23°C in long day photoperiod. (D) Mean and SD of ratios of rosette leaf numbers from the analysis shown in (C). Student’s t-test: *p<0.05; **p≤0.01.DOI: http://dx.doi.org/10.7554/eLife.22114.018