To understand whether the same or different molecular mechanisms are the basis of altered FLM expression in FLM variants, we estimated FLM transcription of variants with strongly altered abundance of processed FLM abundance by measuring levels of unprocessed FLM pre-mRNA from plants grown at 15°C. When compared to the levels of processed FLM mRNA, the PROΔ225bp and INT1Δ373bp variants showed similarly strong reductions of unprocessed pre-mRNA, suggesting that the respective deletion polymorphisms directly affect FLM transcription (Figures 4E,F and and 7A). In turn, the INT6+CAA and PolyA_6xΔ lines had reduced FLM mRNA levels but, when compared to FLMCol-0, did not show substantial changes in unprocessed pre-mRNA levels (Figures 4E,F and and 7B). Since this indicated that post-transcriptional events may be affected in these variants, we tested for the abundance of differential polyadenylated splice variants after semi-quantitative 3’-RACE-PCR and sequencing of the cloned PCR products (Figure 7—figure supplement 1A). There, we detected a relative reduction of FLM-ß transcripts in INT6+CAA and PolyA_6xΔ that was accompanied by increases in the abundance of two polyadenylated transcripts containing exon 1 and intron 1 (E1I1p) that had already been noted in an earlier publication (Figure 7C,D) (Lutz et al., 2015). Importantly, we did not identify a single FLM-δ clone among the 163 sequenced cDNAs. The relative increase in E1I1p transcripts could also be independently confirmed by E1I1p-specific qRT-PCRs and suggested in summary that splicing site choice at the exon 1 - intron 1 junction is changed in the INT6+CAA and PolyA_6xΔ alleles (Figure 7—figure supplement 1B,C). In relation to all exon 1-containing transcripts, the overall abundance of these intron 1-containing transcripts was comparatively low (Figure 7—figure supplement 1C).10.7554/eLife.22114.019Figure 7.FLM polymorphisms affect FLM transcription or splicing at the expense of FLM-ß.(A) and (B) Mean and SD (n = 3) of FLM pre-mRNA levels. Student’s t-test: ***p≤0.001; n.s. = not significant. (C) Schematic representation of FLM cDNAs detected more than once (n = 163). FLM-δ transcripts were not detected and are only shown for completeness. Grey areas correspond to FLM exons of the FLM-ß and FLM-δ gene models as specified in Figure 1A. The arrow indicates the position of the 3' RACE primer used in combination with an oligo(dT) reverse primer to detect polyadenylated transcripts. (D) Frequency distribution of the transcripts displayed in (B) with total number of sequences depicted in the graph.DOI: http://dx.doi.org/10.7554/eLife.22114.01910.7554/eLife.22114.020Figure 7—figure supplement 1.Transgenic lines expressing different FLM alleles display differential FLM expression and splicing patterns.(A) Image of an agarose gel with the results of a 3’-RACE PCR. The primer used for 3’ amplification is indicated in Figure 7C. The gel portions from the FLMCol-0, INT6+CAA and PolyA_6xΔ amplification products framed with a dotted line were purified, cloned and subjected to DNA sequencing. (B) Schematic representation of the primers used for the qRT-PCR quantification of total FLM levels (exon 1) and the E1I1p intron 1 sequence-containing transcripts as indicated by arrows below the gene model. The black box corresponds to FLM exon 1, the grey box to the proximal portion of intron 1. Numbering is respective to the A of the ATG start codon. (C) Mean and SD of qRT-PCR analyses of intron 1 sequence-containing transcripts from four replicate pools of independent T2 lines. The relative decrease in E1I1p expression values compared to exon 1 transcript is indicated for each line in the graph. Note that the comparison of the expression values between the fragments are approximations since the primer efficiencies may not be identical. Primer sequences are listed in Supplementary file 7.DOI: http://dx.doi.org/10.7554/eLife.22114.020