Soybean is an important source of oil, protein, and feed. However, its yield is far below that of major cereal crops. The green revolution increased the yield of cereal crops partially through high‐density planting of lodging‐resistant semi‐dwarf varieties, but required more nitrogen fertilizers, posing an environmental threat. Genes that can improve nitrogen use efficiency need to be integrated into semi‐dwarf varieties to avoid the overuse of fertilizers without the loss of dwarfism. Unlike cereal crops, soybean can assimilate atmospheric nitrogen through symbiotic bacteria. Here, we created new alleles of GmGID1‐2 ( Glycine max GIBBERELLIN INSENSITIVE DWARF 1‐2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease 9 (Cas9) editing, which improved soybean architecture, yield, seed oil content, and nitrogen fixation, by regulation of important pathways and known genes related to branching, lipid metabolism, and nodule symbiosis. GmGID1‐2 knockout reduced plant height, and increased stem diameter and strength, number of branches, nodes on the primary stem, pods, and seeds per plant, leading to an increase in seed weight per plant and yield in soybean. The nodule number, nodule weight, nitrogenase activity, and nitrogen content were also improved in GmGID1‐2 knockout soybean lines, which is novel compared with the semi‐dwarf genes in cereal crops. No loss‐of‐function allele for GmGID1‐2 was identified in soybean germplasm and the edited GmGID1‐2s are superior to the natural alleles, suggesting the GmGID1‐2 knockout mutants generated in this study are valuable genetic resources to further improve soybean yield and seed oil content in future breeding programs. This study illustrates the pleiotropic functions of the GID1 knockout alleles with positive effects on plant architecture, yield, and nitrogen fixation in soybean, which provides a promising strategy toward sustainable agriculture.

Phosphoglycerate kinase (PGK) is a vital glycolytic enzyme that provides energy and carbon skeletons to support fatty acid synthesis. However, the PGK gene family has not been characterized in soybean (Glycine max), and its role in soybean oil accumulation remains unclear. Here, we identified six GmPGK genes in soybean, all of which encode proteins containing conserved PGK domains. Phylogenetic analysis clustered soybean PGK proteins into three groups. Analysis of GmPGK promoters revealed relatively abundant cis-elements related to plant growth, development, and phytohormone response. Expression profiling showed that GmPGK5 transcript abundance increases progressively with oil accumulation during seed development, and is significantly higher in the high-oil variety NN1138-2. Overexpression of GmPGK5 significantly increased total fatty acid content in soybean hairy roots. A single nucleotide polymorphism (SNP) located at Chr15:49447855 within the GmPGK5 promoter was significantly associated with both seed oil content and seed weight in natural soybean accessions. Based on this SNP, a derived cleaved amplified polymorphic sequence (dCAPS) marker was developed to facilitate soybean molecular breeding. Our findings suggest that GmPGK5 may positively regulate fatty acid accumulation in soybean. The identified natural variation and dCAPS marker provide potential valuable tools for marker-assisted selection to improve soybean oil content and seed weight.

First pod height (FPH) is a critical agronomic trait in soybean because it directly affects mechanized harvesting efficiency and harvestable yield. Pods below the cutter-bar height are left uncollected, leading to direct seed loss. However, the genetic architecture underlying FPH remains largely underexplored. Here, we conducted a genome-wide association study (GWAS) on FPH data using 386 soybean accessions evaluated across three environments, together with best linear unbiased prediction (BLUP) values. Phenotypic analysis showed that cultivated soybean exhibited significantly greater FPH than wild accessions, consistent with selection for improved harvestability during domestication. Using two GWAS methods, RTM-GWAS and EMMAX, we identified seven stable (detected by the same method at least three times) quantitative trait nucleotides/loci (QTNs/QTLs) significantly associated with FPH. By integrating sequence variation, gene expression, and functional description, we identified GmAIR9 (corresponding to Glyma.09G084300/SoyZH13_09G078900), which encodes a microtubule-associated protein preferentially expressed in stems, as a candidate gene for further investigation. Haplotype analysis of GmAIR9 revealed that haplotype H3 was associated with low FPH and was detected exclusively in wild soybean accessions, whereas haplotype H1 was associated with increased FPH. Moreover, the frequency of H1 increased from 50.00% in wild soybeans to 71.03% in landraces and 83.47% in improved cultivars. These findings provide valuable genetic resources and promising candidate loci for future validation and breeding applications to optimize FPH and improve the suitability of soybean cultivars for mechanized harvesting.