Fig. 2: Identification and phenotype of gmdmea mutants.

a Generation of mutations in GmDMEa (Glyma.10G202200) by the CRISPR/Cas9 method using single-guide RNA (sgRNA). The sgRNA sequence is shown in red, and the protospacer adjacent motif (PAM) sequence is shown in blue. The coding sequence of GmDMEa that is the same as the sgRNA and its flanking sequences are compared with the corresponding sequences in the gmdmea-1 and gmdmea-3 mutants. The numbers (592 and 614) above the nucleotide sequences are relative to the start codon of GmDMEa. b CAPS markers were used to identify the mutations in the gmdmea mutants. PCR products amplified by specific primers from DN50, gmdmea-1 and gmdmea-3 were digested by SfaN I. The primers used in this experiment are listed in Data 9. M represents the DNA ladder. c Photos of mature seeds harvested from DN50, gmdmea-1 and gmdmea-3 plants to show seed length, width, and thickness. Scale Bar = 1 cm. d Seed size and per-plant yield for DN50, gmdmea-1, and gmdmea-3 genotypes. Seed size data are expressed as mean ± SD from at least 10 individual seeds per biological replicate. The 100-seed weight is also shown as mean ± SD, based on quintuplicate measurements. e Protein and oil composition in dry seeds from DN50, gmdmea-1, and gmdmea-3 plants. Each value represents the mean ± SD of six biological replicates. Pod and branch counts per plant for each line (DN50, gmdmea-1, and gmdmea-3) are also presented. f Pod and branch counts per plant for in DN50, gmdmea-1, and gmdmea-3 lines. Data are mean ± SD for a minimum of nine biological replicates. g Plant height trajectory across reproductive stages R1 to R8. ‘R’ denotes the reproductive phase, with stages R1 to R8 encompassing pod maturation to physiological maturity, culminating in harvest readiness. Displayed are mean values ± SD, with a minimum of nine biological replicates. h–l Complete pods, developing seeds in pods and magnified photo of DN50 and gmdmea-3 seeds at different development stages. DAF, days after flowering. Bar = 1 cm. m Seed dimensional metrics (length, width, and thickness) at multiple developmental phases. Data represent mean ± SD for no fewer than nine biological replicates. All statistical disparities between means were evaluated using unpaired two-tailed t-tests, signifying significance with *p < 0.05 (significant), **p < 0.01 (highly significant), ns p > 0.05 (not significant).