Pseudo-response regulator (PRR) family members serve as key components of the core clock of the circadian clock, and play important roles in photoperiodic flowering, stress tolerance, growth, and the development of plants. In this study, 14 soybean PRR genes were identified, and classified into three groups according to phylogenetic analysis and structural characteristics. Real-time quantitative PCR analysis revealed that 13 GmPRRs exhibited obvious rhythmic expression under long-day (LD) and short-day (SD) conditions, and the expression of 12 GmPRRs was higher under LD in leaves. To evaluate the effects of natural variations in GmPRR alleles on soybean adaptation, we examined the sequences of GmPRRs among 207 varieties collected across China and the US, investigated the flowering phenotypes in six environments, and analyzed the geographical distributions of the major haplotypes. The results showed that a majority of non-synonymous mutations in the coding region were associated with flowering time, and we found that the nonsense mutations resulting in deletion of the CCT domain were related to early flowering. Haplotype analysis demonstrated that the haplotypes associated with early flowering were mostly distributed in Northeast China, while the haplotypes associated with late flowering were mostly cultivated in the lower latitudes of China. Our study of PRR family genes in soybean provides not only an important guide for characterizing the circadian clock-controlled flowering pathway but also a theoretical basis and opportunities to breed varieties with adaptation to specific regions and farming systems.

Flowering time and photoperiod sensitivity are fundamental traits that determine soybean adaptation to a given region or a wide range of geographic environments. The General Regulatory Factors (GRFs), also known as 14-3-3 family, are involved in protein–protein interactions in a phosphorylation-dependent manner, thus regulating ubiquitous biological processes, such as photoperiodic flowering, plant immunity and stress response. In this study, 20 soybean GmSGF14 genes were identified and divided into two categories according to phylogenetic relationships and structural characteristics. Real-time quantitative PCR analysis revealed that GmSGF14g, GmSGF14i, GmSGF14j, GmSGF14k, GmSGF14m and GmSGF14s were highly expressed in all tissues compared to other GmSGF14 genes. In addition, we found that the transcript levels of GmSGF14 family genes in leaves varied significantly under different photoperiodic conditions, indicating that their expression responds to photoperiod. To explore the role of GmSGF14 in the regulation of soybean flowering, the geographical distribution of major haplotypes and their association with flowering time in six environments among 207 soybean germplasms were studied. Haplotype analysis confirmed that the GmSGF14m^H4 harboring a frameshift mutation in the 14-3-3 domain was associated with later flowering. Geographical distribution analysis demonstrated that the haplotypes related to early flowering were frequently found in high-latitude regions, while the haplotypes associated with late flowering were mostly distributed in low-latitude regions of China. Taken together, our results reveal that the GmSGF14 family genes play essential roles in photoperiodic flowering and geographical adaptation of soybean, providing theoretical support for further exploring the function of specific genes in this family and varietal improvement for wide adaptability.

Soybean [Glycine max (L.) Merr.] is a short-day (SD) plant that is sensitive to photoperiod, which influences flowering, maturity, and even adaptation. TEOSINTE-BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors have been shown to regulate photoperiodic flowering. However, the roles of TCPs in SD plants such as soybean, rice, and maize remain largely unknown. In this study, we cloned the GmTCP40 gene from soybean and investigated its expression pattern and function. Compared with wild-type (WT) plants, GmTCP40-overexpression plants flowered earlier under long-day (LD) conditions but not under SD conditions. Consistent with this, the overexpression lines showed upregulation of the flowering-related genes GmFT2a, GmFT2b, GmFT5a, GmFT6, GmAP1a, GmAP1b, GmAP1c, GmSOC1a, GmSOC1b, GmFULa, and GmAG under LD conditions. Further investigation revealed that GmTCP40 binds to the GmAP1a promoter and promotes its expression. Analysis of the GmTCP40 haplotypes and phenotypes of soybean accessions demonstrated that one GmTCP40 haplotype (Hap6) may contribute to delayed flowering at low latitudes. Taken together, our findings provide preliminary insights into the regulation of flowering time by GmTCP40 while laying a foundation for future research on other members of the GmTCP family and for efforts to enhance soybean adaptability.

Expanding soybean planting is vital for food security both in China and globally. The 50° N latitude serves as the northern boundary of major soybean regions. However, enhancing the adaptability of soybean to photothermal conditions enables the potential to extend cultivation to higher latitudes and altitudes. Understanding the genetic basis of super-early maturity of soybean is crucial to achieving this goal. In this study, 438 soybean germplasms collected from high-latitude regions were evaluated in Heihe (HH) (50°15′ N, 127°28′ E, 154 m), Beijicun (BJC) (53°28′ N, 122°21′ E, 295 m) and Labudalin (LBDL) (50°15′ N, 120°19′ E, 577 m). Using resequencing data, we analyzed natural variation and haplotypes in 35 key genes associated with flowering time and maturity. The results showed that the relative maturity groups (RMGs) for BJC, HH, and LBDL were −1.0, 0.0, and −1.2, respectively. Among the 35 genes analyzed, 23 had identical allelic variations, while 12 genes exhibited 19 SNPs and four InDels. Functional mutations were identified in E1, E2, E3, and E4. Notably, all cultivars carried the e1-as allele of E1, which is likely critical for high-latitude adaptation. Additional mutations included a single-base substitution in E2 (16142 A > T) and E3 (5203 C > T), causing premature codon termination, along with frameshift mutations in E4 (3726 and 4099) and E3 (2649). Haplotype analysis revealed significant differences in growth stages among nine gene haplotypes. The higher frequency of early-maturing haplotypes in BJC and LBDL highlights the role of gene accumulation in soybean adaptation. These findings offer valuable insights for improving soybean maturity and expanding its cultivation in high-latitude regions of China.

ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.

Soybean (Glycine max) is a critically important crop for oil, protein, feed, and food security in China. Expanding soybean cultivation into high-latitude regions represents one of the most direct and effective strategies to increase total production. In the present study, we employed KASP (Kompetitive Allele-Specific PCR) marker technology to systematically analyze 18 variant loci across 14 flowering-time genes in 443 soybean germplasm accessions adapted to high-latitude conditions in Arctic Village (Beiji Cun), Mohe City (>53° N), northeastern China. Our results revealed clear functional-tier-dependent selection gradients: key mutation sites (frequency > 96%) in upstream photoreceptors and core circadian clock genes, such as E2 and GmPRR3a, were nearly fixed in the population, whereas downstream flowering genes such as GmFT5b and GmFT2b remained under dynamic selection. Combinatorial analysis of early-maturity allelic variants identified 178 distinct genotype combinations, including six dominant types (n ≥ 10). Field phenotypic analysis demonstrated that the cumulative number of early-maturity alleles was significantly negatively correlated with flowering time, with specific allele combinations such as FT5a^A + FKF1b-hap3^T exhibiting particularly strong flower-promoting effects. A set of 80 highly enriched super-early-maturity accessions, including extreme materials such as MHL22002, were identified, providing valuable genetic resources and a theoretical framework for elucidating the flowering regulatory mechanisms of high-latitude soybean and for breeding super-early-maturing varieties.