To find further non-coding determinants for FLM expression, we analysed FLM nucleotide variation in 776 sequenced Arabidopsis accessions (The 1001 Genomes Consortium, 2016). We identified 45 promoter and intronic SNPs with a minor allele frequency (MAF)≥5% and used these to define ten major haplotypes (H1–-H10) representing 379 (49%) accessions (Figure 3—figure supplement 1A,B; Supplementary file 1). We defined an initial set with 41 accessions by selecting five to twelve accessions from six of the ten haplotype groups (Figure 3A). Since introns 2–6 seemed important for temperature-sensitive FLM expression, we added 11 accessions with varying intron 2–6 haplotypes (HI2-6) but identical intron 1 haplotype (HI1; Figure 3A and Figure 3—figure supplement 1D–G). Finally, we added Col-0 (H3) and Kil-0 (H1) due to their well characterized FLM regulation to obtain a representative FLM haplotype set with ultimately 54 accessions (Supplementary file 2). We assured, by analytical PCR, that none of these accessions, except Kil-0, carried the previously described intron 1 LINE insertion (Lutz et al., 2015).10.7554/eLife.22114.006Figure 3.FLM haplotype analysis of 776 accessions identifies major haplotypes determined by non-coding variation.(A) Haplotype group affiliation of 54 accessions of the FLM haplotype set based on 45 SNPs for either the 7 kb FLM locus (H7kb), intron 1 (HI1) or introns 2–6 (HI2-6). Group numbering and colouring are according to group size. (B) Summarized expression values of total FLM (exon 1), FLM-ß, and FLM-δ expression of the FLM haplotype set. Outliers were determined based on 1.5 x IQR (interquartile range). The coefficient of variation (CV) is shown in the lower graph.DOI: http://dx.doi.org/10.7554/eLife.22114.00610.7554/eLife.22114.007Figure 3—figure supplement 1.Haplotype analysis based on 45 SNPs from 776 accessions.(A) Representation of the position, the frequency and the type of 45 SNPs from 776 accessions used for haplotype clustering. (B), (D), and (E) Haplotype network analysis of the ten (B) most frequent groups of a 7 kb FLM genomic region based on 45 SNPs or the ten (D) or eleven (E) largest haplotype groups when only SNPs of intron 1 (HI1; D) or introns 2–6 (HI2-6; E) are considered. Circle sizes and the color code illustrate number of accessions in each group with group 1 representing 174 (B), 220 (D) and 356 (E) accessions, respectively. In (B), bolt numbers indicate groups from which representative accessions were chosen. (C) Geographic distribution of the ten most frequent groups of a 7 kb FLM genomic region. (F) Venn diagram indicating the overlap of accessions included in the ten largest HI1 and HI2-6 haplotype groups. (G) Clustering of the haplotype groups of the 589 accessions included in the ten largest HI1 and HI2-6 haplotype groups. Accessions were sorted according to their HI1 and HI2-6 haplotype group numbers. Numbers on the left indicate the HI1 haplotype group number as also shown in (D). An enlargement of a subset of accessions highlighted by the dotted boxes is shown on the right. The color coding for haplotype group numbers, which illustrates the group number and group size as shown in (D) and (E) is specified below the graph.DOI: http://dx.doi.org/10.7554/eLife.22114.00710.7554/eLife.22114.008Figure 3—figure supplement 2.Flowering time of the accessions of the FLM haplotype set suggest a vernalization requirement for many accessions.(A) Flowering time analysis of the accessions of the FLM haplotype set. Extremely late flowering accessions or accessions that had not flowered are represented at the 60 rosette leaves margin. Outliers were determined based on 1.5 x IQR (interquartile range). (B) Mean and SD (three biological replicates) in qRT-PCR analyses of FLM-β and FLM-δ expression in twelve day-old Col-0, flc-3 and Col-0 with a FRISf-2FLC vernalization module (Michaels and Amasino, 1999).DOI: http://dx.doi.org/10.7554/eLife.22114.00810.7554/eLife.22114.009Figure 3—figure supplement 3.Correlation analysis identifies FLM-β as the major FLM transcript at 15°C and 23°C in the FLM haplotype set accessions.(A) and (B) Correlation (simple linear regression) between FLM expression as determined by qRT-PCR over FLM exon 1 with measurements of FLM-β and FLM-δ splice variant abundance at 15°C (A) and 23°C (B) in the 54 accessions of the FLM haplotype set. FLM-ß, 15°C, p<0.0001; FLM-δ, 15°C, p=0.009; FLM-ß, 23°C, p<0.0001; FLM-δ, 23°C, p>0.05.DOI: http://dx.doi.org/10.7554/eLife.22114.00910.7554/eLife.22114.010Figure 3—figure supplement 4.119 polymorphic sites among the accessions of the FLM haplotypes set.(A) Position, frequency and type of each the 119 polymorphic sites among 54 accessions of the FLM haplotype set used for simple single locus association tests. (B) Neighbour-joining tree showing the genetic relationship among 54 accessions of the FLM haplotype set based on the 119 polymorphic sites shown in (A). Bootstrap values are indicated at each branch.DOI: http://dx.doi.org/10.7554/eLife.22114.01010.7554/eLife.22114.011Figure 3—figure supplement 5.Results from the simple single locus association test between the 119 polymorphic sites with FLM expression values.(A) and (B) Representation of the –log(10)-transformed p-values of the simple single locus association tests of each of the 119 sites that were included in the analysis. The black horizontal line corresponds to a significance threshold of p=0.05. The black dots represent polymorphic sites that associate with p>0.05, the red dots those with p<0.05. The green dots represent sites with p<0.05 that were chosen for detailed experimental analysis as shown Figure 4A. The FLM genomic gene model is as introduced in Figure 1. Each graph represents the analysis of the indicated expression trait. (A) shows all comparisons with relative expression values and (B) shows all comparisons of relative changes of transcript levels between FLM-β and FLM-δ in response to changes in temperature (15°C to 23°C) or ratios between the transcript levels (FLM-δ/FLM-β) at 15°C and 23°C as a readout for the relative abundance of the two splice forms with hypothesized antagonistic functions. Quantitative values are summarized in Supplementary file 5.DOI: http://dx.doi.org/10.7554/eLife.22114.01110.7554/eLife.22114.012Figure 3—figure supplement 6.Linkage analysis for the simple single locus association test.Pairwise linkage analysis (R2) of the sites used for the simple single locus association test among the 54 accessions of the FLM haplotype set. Only the 109 biallelic sites of the 119 sites are shown. Red circles show the sites PRO1, PRO2, and two biallelic SNPs of the nucleotide triplet INT6 (at bp +3975 and +3976). Position is set to 1 according to the A of the ATG start codon.DOI: http://dx.doi.org/10.7554/eLife.22114.01210.7554/eLife.22114.013Figure 3—figure supplement 7.Effects of the PRO1, PRO2 and INT6 polymorphisms on FLM gene expression.Effects of the PRO1, PRO2 and INT6 alleles on relative expression levels and ratios of these expression values among the 54 accessions of the FLM haplotype set. The background color of each graph indicates that the minor allele associates with upregulation (green) and downregulation (red), respectively. p-values of the association tests are shown: *, p<0.05; **, p≤0.01; ***, p<0.001 as shown in each graph. Non-significant comparisons (p>0.05) are shown with a grey background. Single values are represented as jittered dots. Scale and numbering is set to 1 according to the A of the ATG start codon. Quantitative values are summarized in Supplementary file 5.DOI: http://dx.doi.org/10.7554/eLife.22114.013