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. 2023 Dec 9;22(5):1164–1176. doi: 10.1111/pbi.14254

Flowering time regulator qFT13‐3 involved in soybean adaptation to high latitudes

Yan‐fei Li 1,2,3, † , Liya Zhang 1, † , Jun Wang 4, † , Xing Wang 5, † , Shiyu Guo 1,2, Ze‐jun Xu 5, Delin Li 1,2, Zhangxiong Liu 1,2, Ying‐hui Li 1,2,6,✉, Bin Liu 1,2,6,✉, Li‐juan Qiu 1,2,6,✉
PMCID: PMC11022795  PMID: 38070185

Summary

Soybean is a short‐day plant that typically flowers earlier when exposed to short‐day conditions. However, the identification of genes associated with earlier flowering time but without a yield penalty is rare. In this study, we conducted genome‐wide association studies (GWAS) using two re‐sequencing datasets that included 113 wild soybeans (G. soja) and 1192 cultivated soybeans (G. max), respectively, and simultaneously identified a candidate flowering gene, qFT13‐3, which encodes a protein homologous to the pseudo‐response regulator (PRR) transcription factor. We identified four major haplotypes of qFT13‐3 in the natural population, with haplotype H4 (qFT13‐3 H4 ) being lost during domestication, while qFT13‐3 H1 underwent natural and artificial selection, increasing in proportion from 4.5% in G. soja to 43.8% in landrace and to 81.9% in improve cultivars. Notably, most cultivars harbouring qFT13‐3 H1 were located in high‐latitude regions. Knockout of qFT13‐3 accelerated flowering and maturity time under long‐day conditions, indicating that qFT13‐3 functions as a flowering inhibitor. Our results also showed that qFT13‐3 directly downregulates the expression of GmELF3b‐2 which is a component of the circadian clock evening complex. Field trials revealed that the qft13‐3 mutants shorten the maturity period by 11 days without a concomitant penalty on yield. Collectively, qFT13‐3 can be utilized for the breeding of high‐yield cultivars with a short maturity time suitable for high latitudes.

Keywords: soybean, genome‐wide association study, flowering time, yield‐related traits, adaptation

Introduction

The cultivated soybean (Glycine max (L.) Merr.) can be traced back to its annual wild ancestor, G. soja Sieb & Zucc, which was domesticated approximately 3000–5000 years ago (Qiu and Chang, 2010). As one of the most important agronomic traits, the evolution of flowering time was crucial for the successful domestication and spread of soybeans to adapt to different climatic zones (Nakamichi, 2015).

Soybean is typically considered to be a short‐day crop, with a high degree of photoperiod sensitivity toward flowering. This sensitivity poses a challenge as soybean can hardly mature or flower adequately when planted under natural long‐day conditions. However, natural and artificial selection pressures have given rise to photoperiod insensitive soybean that has spread from East Asia to Europe and North America (Qiu et al., 2011). The determination of long‐day photoperiod insensitivity is primarily affected by various genetic combinations of loss‐of‐functional alleles from flowering repressor loci, such as E1, E2, E3, E4, E7, E8, E10, Tof11, and Tof12, and flowering inducer loci, E9 and J, in soybean (Cober et al., 2010; Cober and Voldeng, 2001; Lu et al., 2017, 2020; Samanfar et al., 2017; Tsubokura et al., 2013; Watanabe et al., 2011; Xu et al., 2013, 2015; Zhao et al., 2016). In spite of the identification of these genes that showcase significant roles in regulating photoperiod sensitivity of flowering, the complete understanding of soybean's adaptation to high latitudes remains lacking.

To address the dual challenges of climate change and farmland reduction, enhancing grain yield presents a crucial pathway toward ensuring global food security. However, the longstanding paradox of ‘high yielding’ and ‘early maturing’ in crop breeding poses a great obstacle to developing cultivars that are both high‐yielding and early maturing. Typically, crops with higher yield require a longer growth period due to the limited carbohydrate production through photosynthesis (Rossi et al., 2015) resulting in a lower yield for crops that mature earlier (Caliskan et al., 2008). In soybean, long‐juvenile gene J shortens flowering and maturity time and reduced overall grain yield (Lu et al., 2017). Consequently, breeders and researchers have invested significant effort in finding solutions to reconcile the trade‐off between ‘early maturing’ and ‘high yielding’ for breeding elite crops for different high latitude regions.

In rice, genetic modifications have been made to enhance nitrogen utilization ability and improve crop yield by manipulating specific genes. Overexpression of OsNRT1.1A/OsNPF6.3 resulted in shorter flowering and maturation times but also produced a higher grain yield, indicating an increase in N utilization ability (Wang et al., 2018). Likewise, the Early flowering‐completely dominant (Ef‐cd) locus in rice has been shown to shorten the maturity duration by 7–20 days without sacrificing yield and enhance nitrogen utilization and photosynthesis rates (Fang et al., 2019). OsDREB1C was found to be an integrator for early flowering, photosynthesis and nitrogen use efficiency which has promising effects in shortening the growth period and boosting grain yields (Wang et al., 2023; Wei et al., 2022). Similarly, the hybrid rice with overexpression of OsNF‐YB4 also exhibited earlier flowering and a shorter growth period with an increase in grain yield attributed to improved photosynthesis (Peng et al., 2023). Notably, unlike rice, no gene has been identified in soybean that can improve grain yield with reduced maturity duration.

PRRs (pseudo‐response regulators) are known to play a crucial role in the feedback loop of the circadian clock and regulate flowering time through photoperiodic pathways (Liang et al., 2021; Wang et al., 2022). The PRR proteins comprise an N‐terminal pseudo‐receiver domain and a C‐terminal CCT domain that facilitate protein–protein and protein‐DNA interactions (Gendron et al., 2012; Nakamichi et al., 2012; Zhai et al., 2022). Past research has confirmed the role of PRR7 in regulating the flowering time in Arabidopsis under long‐day conditions, prompting further investigations in crop plants (Yamamoto et al., 2003). Photoperiod‐H1 (Ppd‐H1) and Photoperiod‐D1a (Ppd‐D1a), the homologues of APRR7 in barley and wheat, respectively, have been found to regulate photoperiod sensitivity and flowering time (Beales et al., 2007; Turner et al., 2005). In the sorghum, SbPRR37 has been found to have a delaying effect on flowering, achieved through the activation of the flowering repressor CONSTANS under long‐day conditions (Murphy et al., 2011). In rice, the OsPRR37 gene was found to exhibit natural variations that contribute to rice cultivation across a range of latitudes. OsPRR37 in the Dongjin background inhibits the expression of Hd3a, thereby suppressing flowering under long‐day conditions. (Koo et al., 2013). However, OsPRR37 in the Zhonghua 11 background promotes flowering by repressing Ghd7 expression under natural long‐day conditions (Hu et al., 2021). In another study, OsPRR73 was found to suppress the expression of Ehd1 by binding to its promoter, which resulted in the inhibiting of heading date in rice (Liang et al., 2021). As for soybean, GmPRR3a/Tof11 and GmPRR3b/Tof12 appear to act through LHYs to promote E1 gene expression and inhibit flowering under long‐day conditions (Li et al., 2020, 2022; Lu et al., 2020; Wang et al., 2020). Despite these findings, it is important to underscore that our current knowledge of the functional properties of PRR genes is still very limited, and more research is necessary to fully characterize PRR gene family in soybean.

In this study, we revealed that the adaption of cultivated soybean to high latitudes is closely associated with a flowering repressor qFT13‐3. This gene was identified through genome‐wide association studies (GWAS) and confirmed through haplotype analysis and gene knock‐out experiments. It was found that qFT13‐3 inhibits the expression of GmELF3b‐2 gene and suppresses flowering in soybean under long‐day conditions. Field experiments showed that the qft13‐3 mutant can significantly reduce the maturity duration without adversely impacting yield under natural long‐day conditions. These findings underscore the significant role of qFT13‐3 as a crucial gene for breeding high‐yield soybean varieties for high latitude regions.

Results

Identification of the qFT13 locus associated with flowering time

To investigate the genetic loci that regulate flower initiation, we conducted genome‐wide association mapping studies (GWAS) on the flowering time trait of cultivated soybean and its wild progenitor G. soja using re‐sequencing data (Li et al., 2023). Our analysis of a panel of 113 G. soja soybeans revealed a locus that contained a peak Single Nucleotide Polymorphism (SNP, Gm13:24984610), which was strongly associated with flowering time in both Beijing (40.1° N, 116.7° E, P value = 2.55E−12) and Jingzhou (30.4° N, 112.2° E, P value = 4.14E−10; Figure 1a,b, Figure S1, Table S1). We also performed GWAS on a panel of 1192 cultivated soybeans (G. max) and identified a strong signal (Gm13:24945777, P value = 1.49E−12) for flowering time in Xuzhou (34.2° N, 117.3° E), which was 38.8 kb away from Gm13:24984610 (Figure S2, Table S2). The linkage disequilibrium block analysis showed a strong linkage between Gm13:24945777 and Gm13:24984610, defining a common QTL region spanning from 24 805 294 to 25 053 917 bp on chromosome 13 (Figure 1c), which we named qFT13 (QTL of flowering time on chromosome 13). Our findings indicated that qFT13 might be the same locus controlling flowering time as reported in previous studies, as it overlapped with three previously reported QTL, including photoperiod sensitivity 5–3 (Liu et al., 2011), reproductive stage length 7–2 (Li et al., 2008), and R8 full maturity 24–2 (Bachlava et al., 2009), and also covered a previously reported SNP (Gm13:24996808) associated with flowering time (Mao et al., 2017).

Figure 1.

Figure 1

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Identification of qFT13‐3 as a flowering time gene by genome‐wide association mapping study. GWAS analysis for flowering time using a panel of 113 G. soja soybeans in Beijing (a) and Jingzhou (b) environments, respectively. (c) Local Manhattan plot (top) and linkage disequilibrium heatmap (bottom) around the peak SNP identified in G. soja and cultivated soybeans. (d) Gene structure (top), four variants (middle), and protein structure (bottom) of four haplotypes of qFT13‐3. (e) The distribution of four haplotypes among G. soja, landrace, and improved cultivar from NR to SR regions. NR, CR, SR, and all indicated north, central, south China, and all accessions, respectively. (f, g) Flowering time variations of haplotypes of qFT13‐3 in G. soja in Beijing and Jingzhou environments and cultivated soybeans in Xuzhou environment. Student's t‐tests were employed to generate P values.

Analysis of genetic variants in the qFT13 locus

Within the qFT13 locus, which contains 22 annotated genes (named qFT13‐1 to qFT13‐22 in Table S3), the genes qFT13‐3 (Glyma.13g135900) and qFT13‐7 (Glyma.13g136300) encode proteins that are orthologous to PSEUDO‐RESPONSE REGULATOR 7 (PRR7) and REVEILLE 8 (RVE8), respectively. These proteins are involved in regulating the circadian clock and flowering time in Arabidopsis. To investigate the genetic variation in these genes, we analysed the genomes of 90 G. soja and 635 cultivated soybean plants that were previously re‐sequenced. We found no nonsynonymous variations or other variants with large effects in qFT13‐7. In contrast, four variants were detected in qFT13‐3, resulting in the formation of four haplotypes (as shown in Figure 1d).

In the G. soja population, all four haplotypes were present, with haplotypes H2 (qFT13‐3 H2 ) and H4 (qFT13‐3 H4 ) being the most prevalent, with frequencies of 42.2% and 34.4%, respectively (Figure 1e). Accessions carrying qFT13‐3 H2 flowered significantly earlier than those carrying qFT13‐3 H4 , as observed in both the Beijing and Jingzhou environments (Figure 1f). Among the four nonsynonymous variants, only Gm13:24842332 showed polymorphism between qFT13‐3 H2 and qFT13‐3 H4 . This suggests that Gm13:24842332 is responsible for the variation in flowering time in G. soja.

In cultivated soybeans consisting of landraces and improved cultivars, all haplotypes of qFT13‐3, except for qFT13‐3 H4 , were identified. The two predominant haplotypes were qFT13‐3 H1 and qFT13‐3 H2 , which were observed at frequencies of 56.7% and 43.0%, respectively (Figure 1e). Accessions carrying qFT13 H1 displayed significantly earlier flowering times than those carrying qFT13 H2 (Figure 1g). Three out of four nonsynonymous variants, except for Gm13:24842332, were polymorphic between qFT13‐3 H1 and qFT13‐3 H2 , suggesting that the combination of Gm13:24844364, Gm13:24842656, and Gm13:24842201 played a significant role in the variation of flowering time in cultivated soybean. These findings suggest that qFT13‐3 is the likely casual gene for the qFT13 locus.

qFT13‐3 H1 contributes to the adaption of cultivated soybean to high latitudes

To investigate the evolutionary patterns of qFT13‐3 under natural and artificial selection, we examined changes in haplotype frequencies in three populations: G. soja, landraces, and improved cultivars. Following domestication, qFT13‐3 H1 , the haplotype associated with early flowering, increased from 4.5% in G. soja to 43.8% (184/420) in landraces, with the concurrent disappearance of qFT13‐3 H4 (Figure 1e). Subsequently, during genetic improvement, the frequency of qFT13‐3 H1 increased further to 81.9% (176/215) in improved cultivars, along with the disappearance of qFT13‐3 H3 (Figure 1e; Table S4). The gradual, successive increase in the prevalence of qFT13‐3 H1 throughout domestication and genetic improvement suggests that it underwent strong artificial selection.

According to the population structure results inferred from neighbour‐joining (NJ) tree, principal components analysis (PCA), and Bayesian clustering (Li et al., 2023), the accessions used in this study were clustered into W_NR, W_CR, W_SR, C_NR, C_CR, C_SR (‘W’ and ‘C’ indicated ‘G. soja’ and ‘Cultivated soybeans (landraces and improved cultivars)’ respectively; NR, CR, and SR indicated north, central, and south China, respectively). Further investigation of cultivated soybeans indicated that the early flowering haplotype qFT13‐3 H1 was almost fixed in the high latitude region (C_NR) and qFT13‐3 H2 was mainly distributed in C_CR and C_SR (Figure 1e), demonstrating that qFT13‐3 H1 played a key role in the adaption of cultivated soybean to high latitudes.

Transcript profiles and subcellular localization of qFT13‐3

To explore the molecular mechanism that underlies the regulation of soybean flowering by qFT13‐3, we conducted quantitative Reverse Transcription PCR (qRT‐PCR) assays to assess the transcript profiles of qFT13‐3 H1 in Williams 82 and qFT13‐3 H2 in Zhongdou 41. The results indicate that both qFT13‐3 H1 and qFT13‐3 H2 are significantly expressed in the trifoliolate leaf, which is the primary tissue responsible for perceiving light signals (Figure 2a), displaying a similar diurnal expression pattern under long‐day conditions, and peaking at 8 h following light illumination (Figure 2b). Notably, qFT13‐3 H1 exhibited higher expression levels in Williams 82 compared to qFT13‐3 H2 in Zhongdou 41. Furthermore, subcellular localization analysis revealed that both qFT13‐3H1 and qFT13‐3H2 proteins were localized in the nucleus (Figure 2c; Figure S4), indicating the possible function of qFT13‐3 as a transcriptional regulator.

Figure 2.

Figure 2

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Expression pattern and subcellular localization of qFT13‐3. (a) Tissue‐specific expression analysis of cultivar Williams 82 and Zhongdou 41. GmActin was used as an internal control with three biological replicates per sample (n = 3). (b) Transcriptional analysis of qFT13‐3 under long‐day at 3 weeks. The second fully expanded trifoliolate leaves were collected every 4 h. (c) Subcellular localization of PA7‐YFP, qFT13‐3H1‐YFP, and qFT13‐3H2‐YFP proteins in Arabidopsis mesophyll protoplasts. PA7‐YFP was used as a control. Scale bars, 20 μm.

CRISPR/Cas9‐induced qft13‐3 mutants display early flowering under long‐day conditions

To investigate the potential involvement of the qFT13‐3 gene in regulating flowering time, we utilized CRISPR/Cas9 technology to create qft13‐3 mutants in the Williams 82 background, using three gRNAs (g1, g2, and g6) designed by CRISPR‐P (http://crispr.hzau.edu.cn; Figure 3a). Through this approach, we identified two homozygous qft13‐3 mutants (qft13‐3‐cr1 and qft13‐3‐cr2), which contained a 1‐bp insertion at g1 and g2 target sites, and a 5‐bp deletion and 1‐bp insertion at the g6 target site, respectively, for further phenotypic analysis (Figure 3b,c). Our results indicate that both qft13‐3‐cr1 and qft13‐3‐cr2 mutants displayed earlier flowering, by 8 days, in comparison to WT Williams 82 plants under long‐day conditions (light/dark = 16 h/8 h), but not under short‐day conditions (light/dark = 12 h/12 h) in a growth chamber (Figure 3d–g), demonstrating that the qFT13‐3 gene functions as a flowering repressor in a long‐day‐dependent manner.

Figure 3.

Figure 3

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Flowering time phenotype of qft13‐3 mutants. (a) Schematic diagram showing the location of mutated sites targeted by gRNAs (g1 and g2 for exon 1, and g6 for exon 4) on the qFT13‐3 genome. (b, c) Sequencing results of target sites in two independent homozygous mutants, qft13‐3‐cr1, and qft13‐3‐cr2, in Williams 82 background. Photographs with indicated lines at the flowering stage and statistical analysis of flowering time under short‐day (d, e) and long‐day conditions (f, g) are presented. Scale bars, 10 cm. The significant difference between the wild‐type Williams 82 and qft13‐3 mutants was determined using Student's t‐tests (***P < 0.001).

qFT13‐3 is a transcriptional repressor of GmELF3b‐2

To investigate the mechanism by which qFT13‐3 inhibits floral initiation, we analysed the expression of various circadian clock genes crucial to the regulation of photoperiodic flowering in soybean, including GmCCA1a, GmCCA1b, GmCCA1c, GmCCA1d, J, GmELF3b‐1, GmELF3b‐2, GmELF4a, GmLUX1, GmLUX2, GmE1, GmE1La, GmE1Lb, GmFT2a, GmFT5a, GmFT4, and GmFT1a by qRT‐PCR (Li et al., 2020; Lin et al., 2021). The results indicated significant upregulation of GmELF3b‐2 transcription levels in qft13‐3 mutants under long‐day conditions (Figure 4c). The patterns and levels of GmCCA1s (Figure S5), GmLUXs (Figure S6), J, GmELF3b‐1, and GmELF4a (Figure 4a,b,d) were comparable between qft13‐3 mutants and WT under long‐day conditions.

Figure 4.

Figure 4

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Analysis of the qFT13‐3 effect on the expression of J, ELF3b‐1, ELF3b‐2, and ELF4a. (a–d) Transcriptional levels of indicated genes during a 24 h cycle under‐long day conditions at 3 weeks, with white and black rectangles representing the light and dark periods, respectively. The second fully expanded trifoliolate leaves were collected every 4 h. Each sample was analysed in triplicate. (e) Diagrams showing the vectors of effector and reporter for dual luciferase assay. (f) Dual‐luciferase assay compared the effect of qFT13‐3H1 and qFT13‐3H2 on ELF3b‐2 Pro::LUC reporter expression. Data are shown as means ± SD for three biological replicates with significant differences indicated by lowercase letters (P < 0.01, ANOVA with Tukey's post hoc test).

We subsequently conducted a Dual‐Luciferase assay to assess whether qFT13‐3 serves as a direct repressor of the GmELF3b‐2 gene. In brief, we examined the transcriptional activities of qFT13‐3H1 and qFT13‐3H2 on the firefly luciferase (LUC) reporter gene, driven by the GmELF3b‐2 promoter, with the Renila reniformis luciferase (REN) gene, driven by the standard 35S promoter, serving as the internal control (Figure 4e). Our findings indicate that both qFT13‐3H1‐Flag and qFT13‐3H2‐Flag significantly decreased the expression of the reporter gene as opposed to the Flag protein alone (Figure 4f). This suggests that qFT13‐3 acts as a transcriptional repressor of GmELF3b‐2 in soybean. Consistent with this, the expression levels of flowering repressors—E1, E1La, E1Lb, GmFT4, and GmFT1a—were significantly reduced, while the expression of flowering activators—GmFT2a and GmFT5a—were notably increased in qft13‐3 mutants under long‐day conditions (refer to Figures S7 and S8). These observations strongly suggest that qFT13‐3 may hinder flowering by influencing the GmELF3b‐2/E1/GmFT pathway in soybean (see Figure S9).

qFT13‐3H1 displays weaker activity than qFT13‐3H2 to repress GmELF3b‐2 expression

To gain an understanding of the differences between qFT13‐3 H1 and qFT13‐3 H2 in regulating flowering, we assessed the correlation between the transcriptional levels of qFT13‐3 and GmELF3b‐2 in soybean accessions carrying haplotype qFT13‐3 H1 and qFT13‐3 H2 under long‐day conditions. For this purpose, we randomly selected 10 accessions harbouring qFT13‐3 H1 and 10 accessions harbouring qFT13‐3 H2 to conduct qRT‐PCR analysis. We measured the expression levels of qFT13‐3 and GmELF3b‐2 in the leaves at two time points (zeitgeber time [ZT] 8 and 12) under long‐day conditions (Figure 5a,b). The observations implied that GmELF3b‐2 expression was comparatively lower in the qFT13‐3 H2 accessions than that in the qFT13‐3 H1 accessions, pointing out that qFT13‐3H2 is more efficient at inhibiting GmELF3b‐2 expression than qFT13‐3H1.

Figure 5.

Figure 5

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Comparison of the effects of qFT13‐3H1 and qFT13‐3H2 on repressing GmELF3b‐2 transcription. (a, b) Correlation scatterplots show the gene expression levels of qFT13‐3 and GmELF3b‐2 genes in different varieties harbouring qFT13‐3 H1 or qFT13‐3 H2 haplotype at ZT 8 and ZT 12 under long‐day conditions, respectively. The transcriptional levels of qFT13‐3 (c, d) and GmELF3b‐2 (e, f) in soybean callus transformed with qFT13‐3 H1 or qFT13‐3 H2 haplotype under long‐day conditions at ZT 8 and at ZT 12, respectively. Violin plots were created using at least five independent calluses for each genotype. Student's t‐tests were used to generate the P values for all comparisons.

Next, we compared the transcriptional repression activities of qFT13‐3H1 and qFT13‐3H2 using the root‐induced callus expression (RICE) system. We produced at least five independent transgenic callus lines for each construct that expressed comparable levels of H1‐Flag or H2‐Flag (Figure 5c,d). The results demonstrated that the expression levels of GmELF3b‐2 in H2‐Flag callus were significantly lower than those in H1‐Flag callus at both ZT 8 and ZT 12 (Figure 5e,f). These findings suggest that qFT13‐3H1 has reduced activity in inhibiting the expression of GmELF3b‐2 relative to qFT13‐3H2, which aligns with the early flowering tendencies of cultivated soybeans carrying qFT13‐3 H1 in comparison to those carrying qFT13‐3 H2 (Figure 1g).

Knockout of qFT13‐3 promotes maturity without reduction in yield

Early maturity usually results in low crop yields because plants need a longer growth period to accumulate carbohydrates via limited photosynthesis (Fang et al., 2019). It is crucial to reduce the association between ‘early maturing’ and ‘low yielding’ for crop breeding. To investigate the potential use of qft13‐3 mutants for breeding cultivars that mature early without yield loss, we conducted a field test at the Shunyi experiment station in Beijing under natural long‐day conditions. The results showed that qft13‐3 mutants flowered and matured, respectively, 3.75 and 11 days earlier than WT plants (Figure 6a‐d), which was consistent with the phenotype when planted in pots under natural long‐day conditions (Figure S10). Next, we compared the yield‐related agronomic traits, such as plant height, node number, 100‐seed weight, and grain weight per plant, between qft13‐3 mutants and WT plants. The results indicate that there was no significant difference in plant height and node number (Figure 6e,f). Notably, the grain weight per plant of qft13‐3 mutants did not change, while the 100‐seed weight increased by 10.6% compared to WT plants (Figure 6h). These findings suggested that qft13‐3 mutants might have the potential to be used for breeding of high‐yield cultivars that mature earlier.

Figure 6.

Figure 6

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Investigation of agronomic traits in qft13‐3 mutants. Representative photos of qft13‐3 mutants and wild‐type plants at the flowering stage (a) and maturity stage (b) under natural‐long day conditions. The plants were sown in early June and harvested in October in Beijing. Scale bar = 10 cm. Statistical analysis of flowering time (c), maturity time (d), plant height (e), node number (f), grain weight per plant (g), and 100‐seed weight (h). Student's t‐tests were used to calculate P values (*P < 0.05; ***P < 0.001).

Discussion

Flowering time is a crucial characteristic for soybean to adapt to different environments (Lin et al., 2021). Identifying new genes that control flowering time is crucial to develop soybean cultivars adapted to specific regions. In regions with higher latitudes, the growing season is relatively short, making early flowering and maturity traits essential. However, the long‐day conditions of higher latitudes delay soybean flowering and maturity, as soybean is a typical short‐day crop. In this study, we identified a haplotype, qFT13‐3 H1 , that is associated with early flowering and CRISPR/Cas9‐induced mutations in the qFT13‐3 gene further accelerate flowering and maturity without yield penalty under natural long‐day conditions, thereby providing potential materials for developing soybean cultivars suitable for higher latitudes.

The qFT13‐3 gene comprises four haplotypes (Figure 1d), which may have undergone fluctuating selection in varied populations (Figure S11). In the G. soja population, all four haplotypes were present, with qFT13‐3 H2 being the most commonly occurring in NR (87.5%) and qFT13‐3 H4 in SR (81.8%), respectively (Figure 1e). The prevalence of each haplotype could be attributed to the fact that accessions carrying qFT13‐3 H2 tend to flower earlier than those carrying qFT13‐3 H4 (Figure 1f), suit for high and low latitudes, respectively. During domestication, qFT13‐3 H4 disappeared, and the frequency of qFT13‐3 H1 increases from 4.5% in G. soja to 43.8% in landraces and 81.9% in improved cultivars (Figure S3). This increase may be due to the early flowering characteristics of accessions carrying qFT13 H1 which blossom even earlier than those carrying qFT13 H2 (Figure 1g). Interestingly, during domestication and improvement, the early flowering qFT13‐3 H1 was strongly selected and nearly fixed in NR. However, its prevalence in landrace reduce to 45.8% and 21.2% in SR and CR respectively. This decrease may be due to the fact that soybean domestication originated in CR (Li et al., 2023), where the early flowering effect of qFT13‐3 H1 possibly impeded the yield increase, leading to selection for qFT13‐3 H2 in CR. Additionally, the short day lengths in SR, combined with a high frequency (72.9%) of summer and autumn soybeans experiencing shorter day lengths, may have contributed to a higher percentage of qFT13‐3 H1 in SR (46.2%) than in CR (20.7%) in cultivated soybeans, as qFT13‐3 H1 has minimal early flowering effect under short‐day conditions.

Cultivated soybeans exhibit lower genetic diversity than their wild counterparts (Lam et al., 2010). Incorporating alleles from wild ancestors can potentially enhance crop adaptation and production. Our study revealed that the late flowering haplotype qFT13‐3 H4 existed in G. soja but was lost in soybean domestication. The qFT13‐3 H4 allele frequency in G. soja increased from 7.1% in NR to 81.8% in SR, implying the significance of qFT13‐3 H4 for G. soja soybean suited to south regions. It would be intriguing to investigate whether introducing the qFT13‐3 H4 allele from G. soja into cultivated soybeans could augment their adaptation to lower latitudes.

We further demonstrated that the CRISPR/Cas9‐induced qft13‐3 mutants display an even earlier maturity without a yield penalty in comparison to the wild‐type Williams 82 harbouring qFT13‐3 H1 . The mutants exhibited earlier maturity, but the plant height and node numbers were same to WT plants. Thus, we speculate that a high growth rate of the qft13‐3 mutants may contribute to the unchanged yield. Usually, early maturing crop plants have a low yield due to a limitation in carbohydrate accumulation. The trade‐off between ‘early maturing’ and ‘high yield’ hinders further improvement in soybean yield. Therefore, our study provides breeders with a valuable genetic resource that is useful for balancing maturity with grain yield for further improving soybean production.

After gene duplication, the gene family underwent functional differentiation (Lan et al., 2009). The RNA‐sequencing data from the SoyBase database showed that the 14 GmPRR genes have similar or diverse expression patterns in soybean, indicating the conservation and differentiation of gene functions (Figure S12). It has been reported that the GmPRR3b gene directly inhibited the expression of GmCCA1a, which further activate J/GmELF3a expression (Li et al., 2020; Lu et al., 2020). Our analysis showed that qFT13‐3 did not affect the expression pattern of GmCCA1s (Figure S5), but directly suppressed the expression of GmELF3b‐2, which is a downstream gene of GmCCA1a (Figure 4; Figures S5 and S9). The distinct downstream regulated genes between qFT13‐3 and GmPRR3b indicate the functional divergence of PRR gene family in soybean, which need to be further explored in future.

Methods

Materials planting and phenotyping

A total of 113 G. soja and 1192 cultivated soybeans were chosen from the Chinese National Soybean GeneBank (Qiu et al., 2013). Three experimental stations whose latitude ranged from 30.4° to 40.1° were used in this study, that is, Beijing (40.1° N, 116.7° E), Jingzhou of Hubei province (30.4° N, 112.2° E), and Xuzhou of Jiangsu Province (34.2° N, 117.3° E). The G. soja panel was evaluated in Beijing and Jingzhou in yield plots of three m2 using three replicates per genotype in 2014. The average values of the three replicates for each location were separately calculated for GWAS analyses. The cultivated panel collected phenotypes over 3 years from 2018 to 2020 in Xuzhou. The best linear unbiased estimator values were calculated for G. soja and cultivated soybeans, respectively, for GWAS analyses. Flowering time was investigated at Beijing, Xuzhou and Jingzhou sites refer to the published criteria (Qiu et al., 2006).

Genome‐wide association study of flowering time

The genome‐wide association studies of 113 G. soja soybeans were conducted using GEMMA (genome‐wide efficient mixed‐model association) by fitting a univariate linear mixed model (Zhou and Stephens, 2012). A panel of 1192 cultivated soybeans was used for a genome‐wide association study using the FarmCPU (Fixed and random model Circulating Probability Unification) model (Liu et al., 2016) implemented in the GAPIT package for R (Liu et al., 2016). After filtering SNPs that minor allele frequency < 5%, the remained dataset was used for principal component analysis. The first five principal components were included as covariates. The P‐value threshold was based on the Bonferroni method (Bland and Altman, 1995) and was calculated using P = 0.05/SNPs.

Plasmid construction and generation of qft13‐3 mutant plants

To generate the qft13‐3 mutant plant, the primer sequences of the qFT13‐3 knockout target site were designed through the CRISPR‐P website (http://crispr.hzau.edu.cn). The GmU6‐sgRNA fragment containing the 19 bp target site sequence was inserted into the 0645‐RPS5A vector at Xbal I site using In‐fusion system. The plasmids were individually introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and then transformed into WT plants (Williams 82) using the cotyledon node method (Paz et al., 2006).

Plant materials and growth conditions

To determine flowering and maturity time, WT (cultivar Williams 82) and mutants were grown in 2021 under long‐day (16 h light/8 h dark) and short‐day (12 h light/12 h dark) conditions in a growth chamber (three replicates, three small pots for each genotype in each replicate, with four plants per pots), and under natural long‐day conditions in Beijing (40.0° N, 116.3° E) that planted on July 2nd and harvested in middle November (three replicates, three big pots for each genotype in each replicate, with seven plants per pots). To further evaluate yield‐related traits, the field test under natural long‐day conditions was performed in Shunyi of Beijing (40.2° N, 116.6° E). Plants were grown on June 10, 2022 with two replicates, a 4 × 2‐m plot for per genotype in each replicate with a plant spacing of 10 cm and a row spacing of 50 cm. After plants were harvested, we analysed the data of grain weight per plant, 100‐seed weight, plant weight, and node number.

RNA extraction, reverse transcription, and quantitative real‐time PCR

To study the expression pattern of qFT13‐3 in Williams 82 and Zhongdou 41, roots, hypocotyls and cotyledons were taken on the fifth day, epicotyls, unifoliate leaf, trifoliolate leaf, stem, and stem tip were taken on the 14th day, samples of the flower were taken after flowering. The plants were grown under long‐day conditions and all samples were collected 8 h after the light was turned on. To study the rhythmic expression of qFT13‐3 H 1 and qFT13‐3 H 2 , after 3 weeks of planting Williams 82 and Zhongdou 41 under long‐day conditions, the second fully expanded trifoliate leaves were taken every 4 h for testing. To explore the dynamic transcription levels of downstream genes in WT plants and qft13‐3 mutant plants, the second fully expanded trifoliate leaves grown under long‐day conditions for 3 weeks were collected every 4 h. To compare the expression levels of GmELF3b‐2 in qFT13‐3 H 1 and qFT13‐3 H 2 accessions, the second fully expanded trifoliate leaves were collected at ZT 8 and ZT 12 for 3 weeks under long‐day conditions. Total RNA was extracted from all the above samples using TRIzol reagent (Invitrogen). For reverse transcription, cDNA was synthesized using a reverse transcription kit (TRAN), and the total RNA amount should not exceed 5 ug. qRT‐PCR was performed by a two‐step method and the calculation formula was 2−▵Ct,▵Ct = CT(gene) − CT(GmActin).

Analysis of subcellular localization

To analyse the subcellular localization of qFT13‐3 H1 and qFT13‐3 H2 , the CDS of qFT13 H1 and qFT13 H2 were inserted into the PA7‐YFP vector at Xmal and BamHI site using In‐fusion system. The PA7‐YFP empty vector was used as a control. Arabidopsis protoplasts were prepared by collecting tender Arabidopsis leaves and incubating them in 10 mL enzyme solution (0.15 g Cellulase R10, 0.04 g Macerozyme R10, 0.728 g D‐Mannitol, 20 m MES pH = 5.7, 20 mm KCl, 10 mm CaCl2) for 3 to 4 h in the dark. Protoplasts were filtered through one layer of 200 mesh screen and pelleted by centrifugation at 100  g for 2 min at 4 °C. Protoplasts were resuspended and washed twice with 10 mL pre‐cooled W5 buffer (154 mm NaCl, 5 mm KCl, 2 mm MES pH = 5.7, 125 mm CaCl2) by centrifuging at 100  g for 2 min at 4 °C, then kept in W5 buffer on ice for 30 min. Protoplasts were collected by centrifugation at 100  g for 2 min at 4 °C and resuspended in an appropriate amount of MMg buffer (0.728 g D‐Mannitol, 15 mm MgCl2, 4 mm MES pH = 5.7). The 10 μL plasmids (1 μg/μL) were mixed with 100 μL Protoplasts and 110 μL PEG (4 g PEG 4000, 0.364 g D‐Mannitol, 0.1 M CaCl2) at room temperature for 5–8 min in the dark. After the reaction, the protoplasts were washed with 440 μL W5 buffer for the first time, then washed twice with 500 μL W5 buffer and finally resuspend the protoplasts in 1 mL W5 buffer at a temperature in the dark. For subcellular localization in tobacco, the CDS of qFT13‐3 H1 and qFT13‐3 H2 were inserted into the PTF101‐GFP vector at XbalIsite using In‐fusion system to generate 35S::H1‐GFP and 35S::H2‐GFP. Then the plasmids were individually introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and then infiltrated into tobacco leaves. The subcellular localization images of the above two assays were captured under a Zeiss LSM780 confocal laser scanning microscope.

Dual‐luciferase reporter assay

To verify the suppression of qFT13‐3 on GmELF3b‐2, the promoter sequence of GmELF3b‐2 was inserted into pGreen0800‐LUC vector digested with restriction enzymes XhoI and BamHI using In‐fusion or T4 system. The CDS of qFT13‐3 H1 and qFT13‐3 H2 were inserted into the 0641‐Flag vector at XhoI site using In‐fusion system to generate H1‐Flag and H2‐Flag. Preparation of Arabidopsis protoplasts using methods in subcellular localization. The plasmids of 0641‐Flag, H1‐Flag, H2‐Flag, and pro‐ELF3b‐2‐LUC were co‐transformed into Arabidopsis protoplasts, respectively. The activities of firefly luciferase (LUC) and Renilla luciferase (REN) were detected using the Dual‐Luciferase® Reporter Assay kit (Promega). The specific process was carried out according to the instructions of the kit. The relative activity of the GmELF3b‐2 promoter was calculated as LUC to REN ratio.

Root‐induced callus expression assay

The plasmids of 0641‐flag, qFT13‐3H1‐Flag, and qFT13‐3H2‐Flag were introduced into Agrobacterium tumefaciens k599 to infect hypocotyl of Williams 82 and induced hair roots growth in root induction medium according to the previously reported method (Kereszt et al., 2007). The hair roots containing 0641‐flag vector served as a control. After 14 days, the hair roots were placed in the callus medium (2.22/L Murashige & Skoog Basal Medium with Vitamins, 0.59 g/L MES monohydrate, 30 g/L sucrose, 1 mg/L 2,4‐D, 0.1 mg/L 6‐BA, 0.1 g/L Timentin, 10 mg/L) about 1 cm to induce callus formation. After the callus grew to an appropriate size, the callus was performed to detect the expression of qFT13‐3 in WT, qFT13‐3 H1 , and qFT13‐3 H2 callus. The H1 and H2 callus with relatively consistent expression levels were selected for subculture. Then, the expression of qFT13‐3 and GmELF3b‐2 were detected in WT, qFT13‐3 H1 , and qFT13‐3 H2 callus under long‐day conditions at ZT 8 and ZT 12. Furthermore, the inhibitory effect of qFT13‐3 H1 and qFT13‐3 H2 on GmELF3b‐2 was analysed.

Funding

This research was supported by the Scientific Innovation 2030 Project (2022ZD0401703), the earmarked fund for CARS (CARS‐04‐PS01), the National Key R&D Program of China (2021YFD1201601), the National Natural Science Foundation of China (U22A20473), the Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences (CAAS‐ZDRW202109), and the Platform of National Crop Germplasm Resources of China.

Conflict of interest

The authors declare that they have no competing interests.

Author contributions

Y.‐F.L., L.Z., Y.‐H.L., B.L., and L.Q. conceived the study and jointly wrote the paper; Y.‐F.L. performed the GWAS and evolutionary analysis; L.Z. conducted the functional experiments; J.W., X.W., Z.X. and S.G. carried out the phenotypic investigation. All authors read and approved the final manuscript.

Supporting information

Figure S1 GWAS analysis for flowering time using a panel of 113 G. soja soybeans with BLUE values.

Figure S2 GWAS analysis for flowering time using a panel of 1305 cultivated soybeans in Xuzhou environment.

Figure S3 The ratio of nucleotide diversity (π) value of three evolutionary populations (G. soja, landrace, and improved cultivar) of genomic regions surrounding qFT13‐3.

Figure S4 Subcellular localization of qFT13‐3H1 and qFT13‐3H2.

Figure S5 The transcriptional level of the GmCCA1 family genes in the WT, qft13‐3‐cr1, and qft13‐3‐cr2 mutants under long‐day conditions at 3 weeks.

Figure S6 The transcriptional levels of the GmLUX family genes in the WT, qft13‐3‐cr1, and qft13‐3‐cr2 mutants under long‐day conditions at 3 weeks.

Figure S7 Investigation of the effects of qft13‐3 mutation on the transcriptional levels of GmE1, GmE1La, and GmE1Lb genes under long‐day conditions at 3 weeks.

Figure S8 Investigation of the effects of qft13‐3 mutation on the expression of the GmFT2a, GmFT5a, GmFT4, and GmFT1a genes under long‐day conditions at 3 weeks.

Figure S9 A proposed model describes the role of qFT13‐3 in soybean flowering time.

Figure S10 Analysis of flowering phenotype of the qft13‐3 mutants under natural long‐day conditions.

Figure S11 A proposed model illustrating the fluctuating selection pressure of qFT13‐3 gene in the different population.

Figure S12 The expression profiles of 14 GmPRRs obtained from the RNA‐seq data deposited in SoyBase (https://www.soybase.org/).

PBI-22-1164-s001.docx (4.1MB, docx)

Table S1 Information of 113 G. soja accessions used for GWAS.

Table S2 Information of 1192 cultivated soybeans used for GWAS.

Table S3 List of candidate genes for flowering time.

Table S4 The 725 accessions carrying four haplotypes of qFT13‐3.

PBI-22-1164-s002.xlsx (95.7KB, xlsx)

Acknowledgements

The authors gratefully acknowledge for ChunMing Xu from Northeast Normal University for these suggestions of evolutionary analysis. This research was supported by Biomedical High Performance Computing Platform, Chinese Academy of Medical Sciences.

Contributor Information

Ying‐hui Li, Email: liyinghui@caas.cn.

Bin Liu, Email: liubin05@caas.cn.

Li‐juan Qiu, Email: qiulijuan@caas.cn.

Data availability statement

All whole genome sequencing data in this study have been deposited in the NCBI Sequence Read Archive under accession number PRJNA681974.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 GWAS analysis for flowering time using a panel of 113 G. soja soybeans with BLUE values.

Figure S2 GWAS analysis for flowering time using a panel of 1305 cultivated soybeans in Xuzhou environment.

Figure S3 The ratio of nucleotide diversity (π) value of three evolutionary populations (G. soja, landrace, and improved cultivar) of genomic regions surrounding qFT13‐3.

Figure S4 Subcellular localization of qFT13‐3H1 and qFT13‐3H2.

Figure S5 The transcriptional level of the GmCCA1 family genes in the WT, qft13‐3‐cr1, and qft13‐3‐cr2 mutants under long‐day conditions at 3 weeks.

Figure S6 The transcriptional levels of the GmLUX family genes in the WT, qft13‐3‐cr1, and qft13‐3‐cr2 mutants under long‐day conditions at 3 weeks.

Figure S7 Investigation of the effects of qft13‐3 mutation on the transcriptional levels of GmE1, GmE1La, and GmE1Lb genes under long‐day conditions at 3 weeks.

Figure S8 Investigation of the effects of qft13‐3 mutation on the expression of the GmFT2a, GmFT5a, GmFT4, and GmFT1a genes under long‐day conditions at 3 weeks.

Figure S9 A proposed model describes the role of qFT13‐3 in soybean flowering time.

Figure S10 Analysis of flowering phenotype of the qft13‐3 mutants under natural long‐day conditions.

Figure S11 A proposed model illustrating the fluctuating selection pressure of qFT13‐3 gene in the different population.

Figure S12 The expression profiles of 14 GmPRRs obtained from the RNA‐seq data deposited in SoyBase (https://www.soybase.org/).

PBI-22-1164-s001.docx (4.1MB, docx)

Table S1 Information of 113 G. soja accessions used for GWAS.

Table S2 Information of 1192 cultivated soybeans used for GWAS.

Table S3 List of candidate genes for flowering time.

Table S4 The 725 accessions carrying four haplotypes of qFT13‐3.

PBI-22-1164-s002.xlsx (95.7KB, xlsx)

Data Availability Statement

All whole genome sequencing data in this study have been deposited in the NCBI Sequence Read Archive under accession number PRJNA681974.

Articles from Plant Biotechnology Journal are provided here courtesy of Society for Experimental Biology (SEB) and the Association of Applied Biologists (AAB) and John Wiley and Sons, Ltd

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