Tianfu Han

SUMMARY: Precise regulation of flowering time and maturity is critical for achieving high and stable soybean yields, and the circadian clock plays a central role in coordinating these developmental transitions. Soybean is a typical short‐day crop with a pronounced photoperiodic response; however, the molecular mechanisms linking circadian regulation to photoperiod‐dependent flowering and maturity remain incompletely understood. Here, we integrated genome‐wide association analysis with deep learning approaches using 257 soybean germplasm accessions spanning 14 maturity groups and identified GmRVE8a , a homolog of the Arabidopsis circadian clock gene REVEILLE , as a strong candidate locus associated with soybean maturity across two field environments. Haplotype analysis demonstrated that accessions carrying the GmRVE8a ^H1 haplotype consistently flowered and matured significantly later than those carrying GmRVE8a ^H2 . Functional validation revealed that knockout of GmRVE8a markedly accelerated flowering and maturity, whereas overexpression of GmRVE8a significantly delayed both processes. qRT‐PCR assays further suggested that GmRVE8a may repress flowering and delay maturity by promoting the expression of GmFT4 , a flowering‐inhibitory member of the FT family, and is unlikely to act by directly regulating E1 expression. Collectively, our findings suggest GmRVE8a as a circadian clock‐associated negative regulator of soybean flowering and maturity, and highlight it as a promising candidate for molecular breeding aimed at improving adaptation to diverse photoperiodic environments.

Photoperiod is a major environmental cue that regulates flowering, maturity, and ecological adaptation in soybean. Although soybean photoperiodic regulation has been extensively studied in true leaves and whole plants, the contribution of cotyledons, the first light-exposed organs after seedling emergence, remains poorly understood. In this study, we used cotyledons of the photoperiod-sensitive soybean cultivar ‘Zigongdongdou’ to investigate early transcriptional responses to day length. Seedlings were grown under short-day conditions, 12 h light/12 h dark, or long-day conditions, 16 h light/8 h dark, and cotyledons were collected every 4 h during the first 72 h after transfer from darkness to photoperiodic conditions, generating a 19-time-point RNA-seq dataset. A total of 663 differentially expressed genes were identified between short-day and long-day conditions, including 340 upregulated and 323 downregulated genes. Functional enrichment analysis showed that these genes were mainly associated with photoperiodic flowering, circadian regulation, hormone signaling, oxidative stress responses, glutathione metabolism, and primary metabolism. Core photoperiod-related genes, including photoreceptors, circadian clock genes, CONSTANS-like genes, E1-family genes, and FT genes, displayed clear photoperiod-dependent expression patterns in cotyledons. Weighted gene co-expression network analysis combined with predicted regulatory network analysis further identified C2H2, WRKY, ERF, NAC, Dof, MYB, and TCP transcription factors as candidate regulators associated with photoperiod-responsive modules. In particular, LATE and several GmNAC genes were connected with flowering- and hormone-related modules, suggesting possible links between transcription factor regulation and the COL–FT pathway. These results demonstrate that soybean cotyledons establish an active photoperiod-responsive transcriptional network shortly after emergence. Cotyledon expression profiling captures major regulatory features of the soybean photoperiod pathway and provides a compact, time-saving, and space-efficient system for investigating early photoperiodic regulation in soybean.

Timely flowering and maturity are crucial for plant reproduction and environmental adaptation. Light–dark cycle-associated regulatory networks integrate photoperiodic cues with intrinsic developmental programs and play pivotal roles in flowering, maturity and environmental adaptation. Soybean is a short-day crop with strong photoperiod responsiveness; however, the molecular mechanisms by which these networks mediate soybean adaptation to diverse environments remain largely elusive. Here, we combine genome-wide association analysis with deep learning-based assessment of soybean maturity variation and identify GmRVE4d, a homolog of the Arabidopsis clock-associated REVEILLE, as a maturity-associated locus across both field environments. Genetic analysis reveals evidence of domestication-related artificial selection at GmRVE4d and identifies the late-maturing haplotype GmRVE4d^H3. Functional analyses further demonstrate that the GmRVE4d protein directly binds to the promoter of GmPRR5a, a soybean homolog of the Arabidopsis clock-associated component PSEUDO-RESPONSE REGULATOR 5, and represses its transcription, thereby delaying soybean flowering and maturity. Taken together, our findings suggest that GmRVE4d is a negative regulator of soybean flowering and maturity that functions by directly repressing GmPRR5a expression and represents a promising molecular target for expanding cultivation latitudes through molecular breeding.

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.

Originating from low and middle latitudes, soybean, common bean, mung bean, and adzuki bean are typical short-day legumes. Through long-term natural evolution and artificial selection, numerous ultra-early maturing varieties adapted to long-day conditions have emerged in these four short-day legumes, becoming a crucial component of agricultural ecosystems in high-latitude regions. Therefore, investigations into the molecular mechanisms governing flowering time and exploration of adaptive mechanisms across diverse short-day legumes are critically essential. In this study, we identified the homologous of soybean key flowering-time genes including E1–E4, GmFT2a, and GmFT5a in common bean, mung bean, and adzuki bean through Blastp. Phylogenetic analysis revealed close genetic relationships, with soybean clustering with common bean, while mung bean and adzuki bean are more closely related. Through cloning of the coding sequences of E1–E4, FT2a, and FT5a homologs in both late and early maturing varieties of soybean, common bean, mung bean, and adzuki bean, natural variations and haplotypes were analyzed and identified. We found that frameshift/nonsynonymous mutations in E1–E4 homologs correlated with early maturity, whereas FT orthologs remained highly conserved in ultra-early maturing varieties of four short-day legumes. Distinct E‑genotype combinations emerged as ecological signatures—ultra-early soybean (e1e2e3e4), common bean (E1e2e3e4), and mung/adzuki beans (e1E2e3e4). Expression levels of E1–E4 in the ultra-early maturing varieties of four short-day legumes were generally lower than in the late maturing varieties, while FT2a/FT5a homologs exhibited the opposite trend. This study reveals the variation hotspots in major flowering genes of short-day legumes and establishes genotype combinations for varieties from different ecological types. These findings provide molecular targets for breeding short-day legume varieties adapted to high-latitude, long-day conditions and offer scientific guidance for expanding the cultivation areas of short-day legumes.

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.