Fig. 2

The Mtphya-1 mutant has reduced sensitivity to far-red light and flowers later than wild type R108 particularly in LD and VLD photoperiods. aMtPHYA with Tnt1 insertions at − 536 bp and − 556 bp upstream of the ATG in the Mtphya-1 (black triangle) and Mtphya-2 (white triangle) mutants, respectively. Exons are black boxes and introns are thin lines. Arrows indicate orientation of Tnt1 insertions. Dotted lines indicate splice sites used in Mtphya-1 compared with R108. b cDNA fragments amplified by primers 3F and 3R. cMtPHYA expression in 14-d-old seedlings in LD, 2 h after dawn, using qRT–PCR with primers 2F and 2R. Mean ± SE three biological replicates, normalized to Medicago PP2A and relative to the highest value. * significantly different expression from R108 using one-way analysis of variance (ANOVA) test between the means (α = 0.05). d-e Seedlings in white light (WL), far-red, FR and dark (d) and ratio of hypocotyl lengths of 3-d-old seedlings to dark-grown (e). Mean ± (t.SE) (0.05), n = 9. (f-g) Flowering time in vernalized LD (VLD) scored as days to flowering (f) or the number of nodes on the primary axis at flowering (g) of the F1 progeny (n = 8) from Mtphya-1 crossed to R108, Mtphya-1 (self-cross) (n = 21), R108 (n = 18) and segregating F2 progeny (n = 217: Mtphya-1 Tnt1 homozygotes, n = 50; heterozygotes, n = 114; wild-type segregants, n = 53) with R108 (n = 25). Data are mean ± (t.SE) (0.05). (h) PCR genotyping fragments from segregating F2 plants in (F-G). Plants were scored as early (e) (like R108) or late (l) flowering relative to R108. 1F and 1R used for wild-type band and 1R and Tnt1F for Tnt1. i-j Graphs showing the flowering time in different conditions of Mtphya-1 mutants (no backcross) and R108 scored in days (i) or nodes to first flower (j). Mean ± (t.SE) (0.05) is presented (n = 9–16)