Haplotype Analysis of GmSGF14 Gene Family Reveals Its Roles in Photoperiodic Flowering and Regional Adaptation of Soybean

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 GmSGF14mH4 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.

The objective of this study was to investigate the molecular and evolutionary characteristics of the soybean GmSGF14 gene family, as well as its role in flowering regulation under different photoperiods. We identified 20 soybean GmSGF14 genes and analyzed their expression patterns under different photoperiodic conditions. Natural variations in GmSGF14 alleles were detected across 207 re-sequenced soybean varieties. Furthermore, we examined the geographical distribution of major haplotypes and their association with flowering time in six different environments. Our findings provided insights into GmSGF14 gene family roles in flowering regulation of soybean.

Identification and Analysis of the Physicochemical Properties of the GmSGF14 Gene Family in Soybean
To identify the GmSGF14 genes in soybean, we performed a BLAST search using the 14-3-3 protein of Arabidopsis as queries via TBtools. Finally, 20 genes were identified and annotated as being GmSGF14 genes based on the complete 14-3-3 domain, before being named as GmSGF14a-GmSGF14t (Supplementary Table S1). The chromosomal localization analysis of GmSGF14 genes showed that they were unevenly distributed in 13 of the 20 soybean chromosomes ( Figure 1A). Further collinearity analysis revealed numerous fragment repetitions among the GmSGF14 gene family, indicating functional similarity among GmSGF14 genes ( Figure 1A). ular weight (MW), the isoelectric point (pI), the instability index and the aliphatic index, were analyzed (Supplementary Table S1). The results showed the GmSGF14 genes encoded proteins with amino acid numbers ranging from 71 aa (GmSGF14t) to 315 aa (GmSGF14k), while MW ranged from 7.92 kDa to 35.2 kDa. The pI of the proteins ranged from 4.67 (GmSGF14g, GmSGF14h) to 5.70 (GmSGF14t), and the instability coefficient varied from 32.3 (GmSGF14t) to 53.8 (GmSGF14r). All of the GmSGF14 proteins were hydrophilic proteins (GRAVY < 0).

Phylogenetic Analysis of the GmSGF14 Gene Family in Soybean
To further explore the evolutionary relationship and classification of the GmSGF14 gene family, a phylogenetic tree was constructed based on the multiple sequence alignment of 14-3-3 protein sequences of Arabidopsis, rice and soybean. The phylogenetic analysis indicated that all the 14-3-3 members were classified into two subfamilies: ε class and non-ε class ( Figure 1B). Among the 20 GmSGF14 proteins, 11 belong to the ε class group (GmSGF14c, d, e, f, l, n, o, p, q, r and t), and 9 belong to non-ε class group (GmSGF14a, b, g, h, i, j, k, m, and s). Furthermore, we found that genes on the same chromosome, such as GmSGF14g and GmSGF14f, and GmSGF14l and GmSGF14m, were not divided into the same group ( Figure 1). These results indicate that GmSGF14 genes experienced evolutionary divergence and functional diversity.

Gene Structure, Motif Composition and Promoter Characterization of the GmSGF14 Gene Family
The exon-intron structure of all the identified GmSGF14 genes was examined to gain more insight into the evolution of the 14-3-3 family in soybean. GmSGF14 genes in the non-ε group had a maximum of four introns, while the number of exons ranged from two (GmSGF14s) to five (GmSGF14k). In the ε group, GmSGF14 genes contained four to six introns and six exons, except for GmSGF14t, which had only one intron and two exons ( Figure 2B). The results indicated that different GmSGF14 genes diverged structurally during evolution. Subsequently, 10 conserved motifs of GmSGF14 were identified through Gene characteristics, including the length of the protein sequence, the protein molecular weight (MW), the isoelectric point (pI), the instability index and the aliphatic index, were analyzed (Supplementary Table S1). The results showed the GmSGF14 genes encoded proteins with amino acid numbers ranging from 71 aa (GmSGF14t) to 315 aa (GmSGF14k), while MW ranged from 7.92 kDa to 35.2 kDa. The pI of the proteins ranged from 4.67 (GmSGF14g, GmSGF14h) to 5.70 (GmSGF14t), and the instability coefficient varied from 32.3 (GmSGF14t) to 53.8 (GmSGF14r). All of the GmSGF14 proteins were hydrophilic proteins (GRAVY < 0).

Phylogenetic Analysis of the GmSGF14 Gene Family in Soybean
To further explore the evolutionary relationship and classification of the GmSGF14 gene family, a phylogenetic tree was constructed based on the multiple sequence alignment of 14-3-3 protein sequences of Arabidopsis, rice and soybean. The phylogenetic analysis indicated that all the 14-3-3 members were classified into two subfamilies: ε class and non-ε class ( Figure 1B). Among the 20 GmSGF14 proteins, 11 belong to the ε class group (GmSGF14c, d, e, f, l, n, o, p, q, r and t), and 9 belong to non-ε class group (GmSGF14a, b, g, h, i, j, k, m, and s). Furthermore, we found that genes on the same chromosome, such as GmSGF14g and GmSGF14f, and GmSGF14l and GmSGF14m, were not divided into the same group ( Figure 1). These results indicate that GmSGF14 genes experienced evolutionary divergence and functional diversity.

Gene Structure, Motif Composition and Promoter Characterization of the GmSGF14 Gene Family
The exon-intron structure of all the identified GmSGF14 genes was examined to gain more insight into the evolution of the 14-3-3 family in soybean. GmSGF14 genes in the non-ε group had a maximum of four introns, while the number of exons ranged from two (GmSGF14s) to five (GmSGF14k). In the ε group, GmSGF14 genes contained four to six introns and six exons, except for GmSGF14t, which had only one intron and two exons ( Figure 2B). The results indicated that different GmSGF14 genes diverged structurally during evolution. Subsequently, 10 conserved motifs of GmSGF14 were identified through Multiple Em for Motif Elicitation (MEME). As shown in Figure 2C, Motif 6 was distributed in all GmSGF14 genes, and seven Motifs (Motif 1-7) constituting the 14-3-3 domain were highly conserved. GmSGF14 members within the same groups were usually found to share a similar motif composition. In addition, motifs 8 and 10 are unique to the non-ε group, and motif 9 is specific to the ε group. Multiple Em for Motif Elicitation (MEME). As shown in Figure 2C, Motif 6 was distributed in all GmSGF14 genes, and seven Motifs (Motif 1-7) constituting the 14-3-3 domain were highly conserved. GmSGF14 members within the same groups were usually found to share a similar motif composition. In addition, motifs 8 and 10 are unique to the non-ε group, and motif 9 is specific to the ε group. In order to explore the potential expression regulation patterns of the GmSGF14 genes, cis-elements were predicted in the 2 kb sequence upstream of these genes (Supplementary Figure S1). A total of 37 cis-acting elements involved in plant growth and development were identified, including elements involved in light response, endosperm expression, cell cycle regulation, meristem expression, circadian rhythm regulation, phytohormone response and stress response (Supplementary Table S2). The number of lightresponse cis-acting elements made up a significant percentage in the promoter regions of 20 GmSGF14 genes. Therefore, we speculated that GmSGF14 may play an important role in growth and development, especially in photoperiodic flowering.

Expression Patterns of GmSGF14 and Its Response to Different Photoperiods
We examined the expression levels of GmSGF14 genes in different tissues (root, hypocotyl, stem, unifoliolate leaf, trifoliolate leaf, and shoot apex) of Zhonghuang 13 (ZH13), which is a soybean variety widely grown in China, under different photoperiod treatments ( Figure 3). Seven genes (GmSGF14h, GmSGF14n, GmSGF14o, GmSGF14p, GmSGF14q, GmSGF14r and GmSGF14t) were hardly expressed in all treatments. In contrast, GmSGF14g, GmSGF14i, GmSGF14j, GmSGF14k, GmSGF14m and GmSGF14s were highly expressed in all tissues. The leaf is the major organ that responds to photoperiod to induce flowering. Thus, we compared the expression of GmSGF14 genes in leaf under LD and SD conditions. We found that under SD conditions, compared with LD conditions, the expression levels of GmSGF14h, GmSGF14m, GmSGF14n, GmSGF14o, GmSGF14p and GmSGF14q were higher in unifoliolate leaves and trifoliolate leaves, while GmSGF14a, GmSGF14b and GmSGF14j showed higher expression only in trifoliolate leaves. Under LD conditions, GmSGF14g and GmSGF14i were highly expressed in the unifoliolate leaves, In order to explore the potential expression regulation patterns of the GmSGF14 genes, cis-elements were predicted in the 2 kb sequence upstream of these genes (Supplementary Figure S1). A total of 37 cis-acting elements involved in plant growth and development were identified, including elements involved in light response, endosperm expression, cell cycle regulation, meristem expression, circadian rhythm regulation, phytohormone response and stress response (Supplementary Table S2). The number of lightresponse cis-acting elements made up a significant percentage in the promoter regions of 20 GmSGF14 genes. Therefore, we speculated that GmSGF14 may play an important role in growth and development, especially in photoperiodic flowering.

Expression Patterns of GmSGF14 and Its Response to Different Photoperiods
We examined the expression levels of GmSGF14 genes in different tissues (root, hypocotyl, stem, unifoliolate leaf, trifoliolate leaf, and shoot apex) of Zhonghuang 13 (ZH13), which is a soybean variety widely grown in China, under different photoperiod treatments (Figure 3). Seven genes (GmSGF14h, GmSGF14n, GmSGF14o, GmSGF14p, GmSGF14q, GmSGF14r and GmSGF14t) were hardly expressed in all treatments. In contrast, GmSGF14g, GmSGF14i, GmSGF14j, GmSGF14k, GmSGF14m and GmSGF14s were highly expressed in all tissues. The leaf is the major organ that responds to photoperiod to induce flowering. Thus, we compared the expression of GmSGF14 genes in leaf under LD and SD conditions. We found that under SD conditions, compared with LD conditions, the expression levels of GmSGF14h, GmSGF14m, GmSGF14n, GmSGF14o, GmSGF14p and GmSGF14q were higher in unifoliolate leaves and trifoliolate leaves, while GmSGF14a, GmSGF14b and GmSGF14j showed higher expression only in trifoliolate leaves. Under LD conditions, GmSGF14g and GmSGF14i were highly expressed in the unifoliolate leaves, and the expression of GmSGF14s was higher in unifoliolate and trifoliolate leaves compared to SD conditions. GmSGF14c, GmSGF14d, GmSGF14e, GmSGF14f, GmSGF14k, GmSGF14l, GmSGF14r and GmSGF14t were more highly expressed in the unifoliolate leaves and less expressed in the trifoliolate leaves under LD conditions compared to SD conditions. These results showed that GmSGF14 genes have different expression patterns and responses to photoperiod, which indicated that GmSGF14 genes might be involved in soybean photoperiodic flowering. and the expression of GmSGF14s was higher in unifoliolate and trifoliolate leaves compared to SD conditions. GmSGF14c, GmSGF14d, GmSGF14e, GmSGF14f, GmSGF14k, GmSGF14l, GmSGF14r and GmSGF14t were more highly expressed in the unifoliolate leaves and less expressed in the trifoliolate leaves under LD conditions compared to SD conditions. These results showed that GmSGF14 genes have different expression patterns and responses to photoperiod, which indicated that GmSGF14 genes might be involved in soybean photoperiodic flowering.

Haplotype Analysis of 20 Soybean GmSGF14 Family Genes in Soybean Germplasm with Diverse Geographical Origins
To evaluate the effect of mutations in GmSGF14 genes on soybean adaptation, we examined the genotypes of 207 re-sequenced soybean varieties. Further geographical distributions regarding major haplotypes and their association with flowering time in six environments were conducted (Figures 4 and 5). In general, haplotypes of the GmSGF14 genes exhibited more diversity in the varieties from China than those from the US, suggesting that the country of origin for cultivated soybean is China ( Figure 5).    Based on 14 SNPs and 4 Indels, GmSGF14c was divided into 6 haplotypes (Supplementary Figure S2C). GmSGF14c H1 showed earlier flowering, and the frequency of GmSGF14c H1 decreased with decreasing latitude in China (Figures 4C and 5C). With the increase in latitude in China, the proportion of GmSGF14c H3 increased ( Figure 5C).
Five haplotypes were identified based on seven SNPs and five Indels of GmSGF14f (Supplementary Figure S2E). It was found that GmSGF14f H1 was mostly distributed in NE and the US, with significantly earlier flowering compared with GmSGF14f H2 , GmSGF14f H3 and GmSGF14f H4 (Figures 4E and 5E). GmSGF14f H5 showed significantly later flowering among plants distributed in HHH and SC than those found in the NE and the US ( Figures  4E and 5E). Further analysis of these two unique haplotypes (GmSGF14f H1 and GmSGF14f H5 ) may contribute to the genetic improvement of soybean in SC and NE.
Three SNPs and three haplotypes were identified for GmSGF14h ( Supplementary Figure S2F). GmSGF14h H2 was the haplotype most widely distributed across China and the  (Figures 4A and 5A). The results indicated that GmSGF14a H5 could better adapt to short-day environments and might facilitate soybean genetic improvement in low-latitude regions such as Southern China (SC).
Four SNPs were located in the 5'UTR and intron regions, and three haplotypes were discovered for GmSGF14b (Supplementary Figure S2B). GmSGF14b H3 was distributed in Huang-Huai-Hai Rivers Valley Region (HHH) and Northeastern China (NE), while GmSGF14b H2 covered a large proportion of the haplotypes found across China and the US. GmSGF14b H3 exhibited significantly later flowering in Heihe; however, it showed no significance in the five other environments (Figures 4B and 5B).
Based on 14 SNPs and 4 Indels, GmSGF14c was divided into 6 haplotypes (Supplementary Figure S2C). GmSGF14c H1 showed earlier flowering, and the frequency of GmSGF14c H1 decreased with decreasing latitude in China (Figures 4C and 5C). With the increase in latitude in China, the proportion of GmSGF14c H3 increased ( Figure 5C).
Five haplotypes were identified based on seven SNPs and five Indels of GmSGF14f (Supplementary Figure S2E). It was found that GmSGF14f H1 was mostly distributed in NE and the US, with significantly earlier flowering compared with GmSGF14f H2 , GmSGF14f H3 and GmSGF14f H4 (Figures 4E and 5E). GmSGF14f H5 showed significantly later flowering among plants distributed in HHH and SC than those found in the NE and the US (Figures 4E and 5E). Further analysis of these two unique haplotypes (GmSGF14f H1 and GmSGF14f H5 ) may contribute to the genetic improvement of soybean in SC and NE.
Three SNPs and three haplotypes were identified for GmSGF14h (Supplementary Figure S2F). GmSGF14h H2 was the haplotype most widely distributed across China and the US (Figure 5F), and there was no significant difference in flowering times among the three haplotypes ( Figure 4F).
We found one SNP and two Indels in GmSGF14i, and identified three haplotypes (Supplementary Figure S2G). Among the three haplotypes, GmSGF14i H1 associated with significantly earlier flowering was mainly distributed in NE and the US. GmSGF14i H2 was the most abundant ( Figure 5G), while GmSGF14i H3 flowered later and tended to distribute in HHH and SC ( Figures 4G and 5G).
We found 47 SNPs and two haplotypes in GmSGF14k (Supplementary Figure S2H). In six environments, GmSGF14k H2 only showed significantly earlier flowering in Xinxiang compared to GmSGF14k H1 ( Figure 4H) and was the most widely distributed haplotype across China and the US ( Figure 5H).
For GmSGF14m, a total of 4 haplotypes were identified based on 10 SNPs and 6 Indels (Supplementary Figure S2I). GmSGF14m H4 carried a frameshift mutation that resulted in partial deletion of 14-3-3 domain in the encoded protein (Supplementary Figure S2I). Compared to GmSGF14m H1 , GmSGF14m H4 showed significantly later flowering and the percentage of GmSGF14m H1 increased with the increase in latitude in China, while GmSGF14m H4 was had the opposite characteristics ( Figures 4I and 5I). These results suggest that the frameshift mutation carried out in GmSGF14m H4 leading to the loss of 14-3-3 domain may result in late flowering.
Based on six SNPs, two haplotypes were identified for GmSGF14n (Supplementary Figure S2J). GmSGF14n H1 , which was primarily distributed at high latitudes, flowered significantly earlier than GmSGF14n H2 (Figures 4J and 5J). The percentage of GmSGF14n H2 increased with the decrease in latitude in China ( Figure 5J).
One SNP and one Indel were found in GmSGF14o, and three haplotypes were identified (Supplementary Figure S2K). Through comparing the flowering time of the three haplotypes in the six environments in China, we found that GmSGF14o H3 flowered significantly later (except Sanya and Xiangtan), and the phenotypic difference tended to become more pronounced with increasing latitude ( Figure 4K). The frequencies of GmSGF14o H3 varied across regions, and no variety found in the US harbored this haplotype ( Figure 5K).
Based on 12 SNPs and 4 Indels, GmSGF14q was divided into 3 haplotypes (Supplementary Figure S2M). The percentage of GmSGF14q H1 was high in NE, and GmSGF14q H2 and GmSGF14q H3 showed later flowering and were mainly found in HHH and SC ( Figures 4M and 5M).
For GmSGF14r, 25 SNPs and 3 Indels were discovered, and SNP-Chr20:2850044 was located at the 14-3-3 domain, resulting in an amino acid substitution (Glu/Asp) (Supplementary Figure S2O). Eight haplotypes were identified and were related to different genetic effects on flowering under different environments ( Figure 4N). GmSGF14r H4 was the haplotype most widely distributed across China and the US ( Figure 5N). The rich genetic diversity of GmSGF14r may contribute to the varied phenotypic effects on soybean flowering across different photoperiods.
Among the five SNPs in GmSGF14s, SNP-Chr17:34108428, SNP-Chr17:34108738 and SNP-Chr17:34108786 were missense mutations that resulted in amino acid substitutions (Supplementary Figure S2N). We identified two haplotypes, of which GmSGF14s H2 was mainly distributed in HHH and only showed significantly later flowering in Heihe when compared to the other five environments. (Figures 4O and 5O).

Discussion
In plants, previous studies showed that 14-3-3 proteins are involved in various biological processes, including photoperiodic flowering [44,45,55], plant immunity [55,56] and stress responses [57,58]. However, comprehensive analysis of the 14-3-3 family and their functions in soybean flowering is limited. In this study, a total of 20 GmSGF14 genes were identified and classified into ε and non-ε classes via phylogenetic analysis ( Figure 1B), which was consistent with model plants, such as Arabidopsis [59][60][61] and rice [62]. This discovery demonstrated that 14-3-3 genes experienced expansion in soybean, compared to 15 genes in Arabidopsis and 8 genes in rice. Chromosomal distribution and synteny analysis confirmed that gene and segmental duplication events played important roles in the expansion of GmSGF14 gene family ( Figure 1A), supporting the complex history of whole genome duplications in soybean [26,63,64]. Conserved protein motifs analysis further demonstrated that ε and non-ε groups possess different motif structures (Figure 2), indicating diverse functions in accordance with 14-3-3 proteins in Arabidopsis and rice. Expression profiles revealed that soybean GmSGF14 genes in all examined organs showed differential expression, and their expression in leaf and shoot apex varied significantly under different light conditions, suggesting their involvement in photoperiodic regulation of soybean flowering (Figure 3).
The cultivated soybean originated in the temperate region of China and was planted worldwide as an important economic crop due to its high protein and oil content [66]. This wide distribution can be attributed to the rich natural variations in and combinations of genes controlling flowering times [67,68]. Natural variation in GmELF3 confers long juvenility and improves soybean adaptation in the tropics [19]. In contrast, natural variations in GmPRR37 (GmPRR3b) affect photoperiodic flowering and contribute to soybean adaptation in high-latitude regions [24,25]. To evaluate the effects of variations in GmSGF14 on soybean flowering and adaptation, we investigated the genotypes of 207 vari-eties collected across China and the US, and analyzed the flowering-time phenotypes and geographical distributions of the major haplotypes. We found that GmSGF14m H4 carries a single-base deletion that results in a frameshift mutation and premature termination of the encoded protein (Supplementary Figure S2I). This null mutant was significantly associated with late flowering, and its frequency increased with decreasing latitude in China (Figures 4I and 5I). These results indicate that GmSGF14m might function as a flowering promoter, while the frameshift mutation may lead to late flowering. Additionally, we found that GmSGF14c H1 , GmSGF14e H1 , GmSGF14f H1 , GmSGF14i H1 , GmSGF14m H1 , GmSGF14n H1 , GmSGF14p H1 , GmSGF14p H2 , GmSGF14q H1 and GmSGF14r H2 were associated with early flowering and primarily distributed in higher latitudes of China. On the other hand, GmSGF14a H5 , GmSGF14e H2 , GmSGF14e H3 , GmSGF14f H5 , GmSGF14i H3 , GmSGF14m H4 , GmSGF14n H2 , GmSGF14o H3 , GmSGF14p H3 , GmSGF14q H3 and GmSGF14r H5 were related to late flowering and primarily distributed in lower latitude regions. We speculate that the diverse genetic variation in GmSGF14 contributed to soybean cultivation across different latitudes. Developing Kompetitive Allele Specific PCR (KASP) markers and gene chips for these variations can provide information about the molecular breeding of soybean flowering times. Further functional characterization of GmSGF14 family genes in soybean could provide opportunities to utilize genome-editing tools to modify the functional status of GmSGF14 family members, thus facilitating precise prediction of flowering times in soybean genetic improvement.

Plant Materials, Treatments and Multiple-Site Experiments
For the expression pattern analysis of GmSGF14 family genes, a widely grown soybean variety Zhonghuang 13 (ZH13) was grown in a controlled culture room at 26 • C under short-day (SD, 12 h: 12 h, light: dark) and long-day (LD, 16 h: 8 h, light: dark) conditions. After entraining for 14 days, root, hypocotyl, stem, unifoliolate leaf, trifoliolate leaf and shoot apex were sampled. Samples were collected after 4 h exposure to light.
A total of 207 re-sequenced soybean germplasms were used for haplotype analysis [ [17]. Flowering times for 207 soybean varieties in six environments were recorded as days from the emergence to the R1 stage (the time at which the first flower opens at any node on the main stem) [70] and determined through taking the average of two replicates.
Duplications of GmSGF14 family genes were analyzed via the Multiple Collinearity Scan toolkit (MCScanX) with the default parameters, and a visual synchronous analysis diagram was constructed using TBtools [71]. The amino acid numbers, molecular weight (MW), isoelectric point (pI), instability coefficient, fat coefficient and hydrophilicity of identified GmSGF14 proteins were analyzed via ExPASy ProtParam (https://web.expasy. org/protparam/) (accessed on 28 September 2022). MEGA 7.0 software was used to perform multiple sequence alignment on the reported 14-3-3 protein sequences of Arabidopsis thaliana, rice and soybean, as well as to construct the phylogenetic tree with the Neighbor-Joining (NJ) method. The bootstrap value was set to 1000. Bootstrap resampling (100) was used to assess the reliability of interior branches, and other parameters were default values.

Sequence Analysis of Soybean GmSGF14 Family Genes
The exon-intron pattern of GmSGF14 gene family was analyzed using TBtools v1.0+ software through inputting gene annotation GFF files. The online program MEME (https: //meme-suite.org/meme/tools/meme) (accessed on 9 October 2022) was used to identify the conserved motif of GmSGF14 proteins, and the maximum number of motifs was set to 10. To identify the cis-elements, TBtools was used to extract a 2 kb genomic sequence upstream from the start codon (ATG) of the GmSGF14 family genes gathered from the soybean genome database, and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/) (accessed on 21 October 2022) was used to predict the cis-acting elements of the promoter. Finally, TBtools v1.0+ software was used for visual mapping [71].

Expression Profile Analysis of Soybean GmSGF14 Gene Family
The total RNA of different tissues of ZH13 was extracted using Easy Fast Plant Tissue Kit (TianGen, Beijing, China), and RNA was reverse transcribed into cDNA with the FastKing RT Kit (With gDNase) (TianGen, Beijing, China). Primers for qRT-PCR were designed using the NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed on 14 December 2022) (Supplementary Table S4). Using ABI QuantStudio TM 7 flex (Applied Biosystems, San Francisco, CA, USA), qRT-PCR was performed with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), and each sample contained three biological replicates. GmActin (Glyma18g52780) was used as the internal reference, and the relative expression was calculated via the 2 −∆∆Ct method.

Haplotype and Correlation Analysis of Soybean GmSGF14 Gene Family
The natural variation in GmSGF14 genes was retrieved from the NCBI database under Short Read Archive (SRA) Accession Number SRP062560 and PRJNA589345 [17,69], and the haplotype and data processing analyses were performed using TASSEL 5 and EXCEL. We used GraphPad Prism 8 to analyze the association between GmSGF14 haplotypes and flowering time via Duncan's multiple range test with p < 0.05 as the significant level.