Supplementary Figure 1 The phenotypes of PI 159925, BR121, and Harosoy under short-day conditions. (a) Plant height. (b) Number of branches. (c) Average internode length. (d) Number of nodes. (e) Pods per plant. (f) Grains per plant. The plants were grown in a standard field with artificially controlled short-day conditions (12 h light/12 h dark). All data are given as means ± s.e.m. (n = 10 plants). One-tailed, two-sample t-tests were used to generate the P values.
Supplementary Figure 2 QTL mapping of the J locus. (a,b) Whole-chromosome scan of QTLs in two F 2 populations, PI 159925 Harosoy (a) and BR121 Harosoy (b). Red lines indicate the threshold for QTL detection. Detailed QTL information is provided in Supplementary Table 1. QTLs were evaluated for flowering time under short-day conditions for the two F 2 populations. (c,d) Allelic effects on flowering time of QTLs of J and E1 in two F 2 populations, PI 159925 Harosoy (c) and BR121 Harosoy (d). Allelic combinations of the J and E1 loci are indicated in each column. The numbers correspond to the plants tested for each allelic combination of J and E1. Genotyping of J and E1 is described in the Online Methods.
Supplementary Figure 3 Protein sequence alignment of loss-of-function j alleles and nonsynonymous SNPs. The amino acid sequence in the box is the binding position for the antibody to J. A red arrowhead indicates the critical amino acid substitutions that reduced the functions of J, as confirmed by transient assays with the E1 promoter (Supplementary Fig. 10c). Sequences were aligned with Clustal X.
Supplementary Figure 4 Phenotypes of the two transformants and of BR121 and the NILs of J under short-day conditions. (a f) Plant height (a), average internode length (b), number of branches(c), number of nodes (d), pods per plant (e), and grains per plant (f) in the two transformants and BR121. (g j) Plant height (g), number of nodes (h), pods per plant (i), and grains per plant (j) in NILs of J. All plants were grown in a standard field with artificially controlled short-day conditions (12 h light/12 h dark). All data are given as means ± s.e.m. (n = 10 plants). One-tailed, two-sample t-tests were used to generate the P values.
Supplementary Figure 5 The genotypes of the NILs of J and E1 derived from the cross between BR121 and Harosoy. (a) Molecular markers and their genomic positions on chromosome 4 (top) and chromosome 6 (bottom). (b) Introgression segments of J on chromosome 4 (top) and E1 on chromosome 6 (bottom) in NIL-J/j. (c) Introgression segments of J on chromosome 4 (top) and E1 on chromosome 6 (bottom) in four NILs segregating at the E1 and J loci. White represents the segments derived from BR121, and gray represents the segments derived from Harosoy. Squares indicate the different homozygous introgression segments for the J and E1 loci. Primers are listed in Supplementary Table 9.
Supplementary Figure 6 Phylogenetic tree of J and its complementation in the Arabidopsis elf3-8 mutant. (a) Phylogenetic tree of J and ELF3 proteins from different species. (b) Complementation of flowering of the Arabidopsis elf3-8 mutant by pelf3:j and overexpression of p35s:j in wild-type Col-0. Scale bar, 10 cm. (c) Flowering time corresponding to rosette leaf numbers from the plants in b. All transformants were from Arabidopsis T 1 transgenic lines.
Supplementary Figure 7 Diurnal expressions of J, E1, FT2a, and FT5a in transformant TC#2 and BR121 and in NILs of J under short-day conditions. Plants were grown until 20 DAE. Each sample was collected from three plants and bulked. qrt PCR results were from three technical replicates and are shown as means ± s.e.m.
Supplementary Figure 8 Diurnal expressions of J and E1 in NILs of E1 and E3 E4, and flowering response under short-day conditions. (a) Diurnal expressions of J in NILs of E1. (b,c) Diurnal expression of J (b) and E1 (c) in NILs of E3 E4. (d) Flowering time of NILs of E3 E4. Plants were grown until 20 DAE. Each sample was collected from three plants and bulked. qrt PCR results are from three technical replicates and are shown as means ± s.e.m. Flowering time was recoded from 15 plants for each NIL. One-tailed, two-sample t-tests were used to generate the P values.
Supplementary Figure 9 Specific test of J antibody. Leaf proteins of 20-d-old TC#2 (pj:j) and BR121 (j-2) seedlings grown under short-day conditions were tested by immunoblot with J antibody. An asterisk denotes the specific band of J protein, and a star indicates the unspecific band that can act as a loading control. The image of the immunoblot is full length.
Supplementary Figure 10 GUS activity with mutation of the GATWCG motifs in the E1 promoter and GUS activity of J proteins with a nonsynonymous SNP on suppression of the E1 promoter in Arabidopsis transient assays. (a) Sequence motifs of the LBS binding sites in the E1 promoter. Red indicates the native sequences used for the pe1-gus construct. Blue indicates the mutant sequences used for the mpe1-gus construct. (b) The relative GUS activity of the E1 promoter is suppressed by J protein. GUS activity is from three independent replicates and is shown as means ± s.e.m. (c) The relative GUS activity of J proteins with a nonsynonymous SNP on suppression of the E1 promoter. The presence of different lowercase letters above the histogram bars denotes significant differences across the two panels (P < 0.05). GUS activity is from six independent replicates and is shown as means ± s.e.m. One-tailed, two-sample t-tests were used to generate the P values.
Supplementary Figure 11 Flowering-time response of J under long-day (14 h light/10 h dark) conditions. (a,b) Flowering time of transformants TC#2 (a) and TC#6 (b) versus BR121 under long-day conditions. All data are given as means ± s.e.m. (n = 10 plants). One-tailed, two-sample t-tests were used to generate the P values. (c f) Diurnal expression of J (c), E1 (d), FT2a (e), and FT5a (f) in transformant TC#2 versus BR121 under long-day conditions. Plants were grown until 20 DAE. Each sample was collected from three plants and bulked. qrt PCR results are from three technical replicates and are shown as means ± s.e.m.
Supplementary Figure 12 Proposed model of the photoperiod-regulated flowering pathway under short-day conditions. J is suppressed by two PHYAs, E3 and E4, and the J protein physically binds to the promoter of E1 near the LUX-binding motif to suppress E1 transcription. This relieves the E1-dependent transcriptional repression of FT2a and FT5a, which promote flowering. When the function of the J gene is impaired, E1 itself is released from repression and is able to repress FT2a and FT5a, resulting in later flowering.
Supplementary Figure 13 Flowering time variations of 15 accessions harboring mutant alleles of J under short-day conditions. Details of the 15 accessions are provided in Supplementary Table 6. The plants were grown in a standard field with artificially controlled short-day conditions (12 h light/12 h dark). All data are given as means ± s.e.m. (n = 5 plants)
Supplementary Figure 14 Mutant alleles of J and phylogenetic tree of 302 resequenced soybean accessions. The 302 resequenced soybean accessions represent the process of soybean domestication and improvement. Dark gray clusters represent wild soybeans, violet clusters represent landraces, and blue clusters represent improved cultivars. Blue circles correspond to the normal allele and red circles correspond to mutant alleles of J.
Supplementary Figure 15 Phenotypes of Guizao 1 versus Huaxia 3 and Huaxia 3 (j-4) versus its complementation transgenic T 4 line TC#H7 (pj:j) under short-day conditions. (a) Huaxia 3 had increased plant height, node numbers, and pod numbers. Scale bar, 10 cm. (b g) Plant height (b), number of nodes (c), number of branches (d), pods per plant (e), grains per plant (f), and yield per plant (g) of Guizao 1 and Huaxia 3. (h o) Flowering time (h), maturity (i), plant height (j), number of nodes (k), average internode length (l), number of nodes (m), pods per plant (n), and grain yield per plant (o) in Huaxia 3 and transgenic line TC#H7. The plants were grown in a standard field with artificially controlled short-day conditions (12 h light/12 h dark). All data are given as means ± s.e.m. (n = 10 plants). One-tailed, two-sample t-tests were used to generate the P values.