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. 2019 Jul 5;18(1):298–309. doi: 10.1111/pbi.13199

Mutagenesis of GmFT2a and GmFT5a mediated by CRISPR/Cas9 contributes for expanding the regional adaptability of soybean

Yupeng Cai 1,2,†, Liwei Wang 2,†, Li Chen 1,2,†, Tingting Wu 2, Luping Liu 2, Shi Sun 2, Cunxiang Wu 2, Weiwei Yao 1,2, Bingjun Jiang 2, Shan Yuan 2, Tianfu Han 2,✉, Wensheng Hou 1,2,✉
PMCID: PMC6920152  PMID: 31240772

Summary

Flowering time is a key agronomic trait that directly influences the successful adaptation of soybean (Glycine max) to diverse latitudes and farming systems. GmFT2a and GmFT5a have been extensively identified as flowering activators and integrators in soybean. Here, we identified two quantitative trait loci (QTLs) regions harbouring GmFT2a and GmFT5a, respectively, associated with different genetic effects on flowering under different photoperiods. We analysed the flowering time of transgenic plants overexpressing GmFT2a or GmFT5a, ft2a mutants, ft5a mutants and ft2aft5a double mutants under long‐day (LD) and short‐day (SD) conditions. We confirmed that GmFT2a and GmFT5a are not redundant, they collectively regulate flowering time, and the effect of GmFT2a is more prominent than that of GmFT5a under SD conditions whereas GmFT5a has more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions. The ft2aft5a double mutants showed late flowering by about 31.3 days under SD conditions and produced significantly increased numbers of pods and seeds per plant compared to the wild type. We speculate that these mutants may have enormous yield potential for the tropics. In addition, we examined the sequences of these two loci in 202 soybean accessions and investigated the flowering phenotypes, geographical distributions and maturity groups within major haplotypes. These results will contribute to soybean breeding and regional adaptability.

Keywords: soybean, CRISPR/Cas9, GmFT2a, GmFT5a, flowering time, regional adaptability

Introduction

Soybean (Glycine max (L.) Merr.) originated in the temperate regions of China between 32.0°N and 40.5°N (Li et al., 2008b), and has been introduced as a crop plant to Korea, Japan and many countries in North/Central/South America. It has become one of the most important economic crops on account of its high oil and protein concentrations (Wilson, 2008). A major factor for the distribution of soybean cultivation across a wide range of geographical regions, and responsible for large impacts on yield, is its diversity in flowering time. Floral transition is synchronized by the integration of signals from factors that are both endogenous (such as gene, plant age and hormone status) and environmental (such as photoperiod and temperature) (Song et al., 2013). Among the various environmental signals, photoperiod is one of the major determinants of soybean's adaptation to seasonal changes in day length for flowering (Bäurle and Dean, 2006). Soybean is known as a short‐day plant that accelerates transition from the vegetative phase to the reproductive phase when it senses short‐day (SD) conditions. Soybean still flowers under long‐day (LD) conditions, albeit much later than in SD conditions. Thus, investigating the responses of flowering to photoperiod has great significance in soybean regional introduction and domestication (Wang et al., 2016).

In the model plant Arabidopsis thaliana, at least four major flowering pathways, namely the vernalization, autonomous, gibberellin (GA) and photoperiod pathways, are known to regulate the floral transition process (Fornara et al., 2010). These four pathways regulate the expression of FLOWERLOCUST (FT), which plays an important role in flowering pathways as an integrator (Turck et al., 2008) and encodes a florigen protein that is transported from leaves to shoot apical meristems (SAMs) through the phloem and that functions as a long‐distance signal to induce floral initiation (Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007; Notaguchi et al., 2008). FT and FD (a bZIP transcription factor) are interdependent partners through protein interaction and act at the SAMs to promote floral transition through transcriptional activation of the floral meristem identity gene APETALA1 (AP1) (Abe et al., 2005; Wigge et al., 2005). Ectopic expression of ArabidopsisFT in soybean induced precocious flowering under non‐inductive conditions (Yamagishi and Yoshikawa, 2011), providing evidence that FT promotes flowering in soybean as well.

Ten FT homologs have been identified in soybean. Among them, GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) have been confirmed to have main promoting effects on flowering time (Kong et al., 2010). At present, there are three models, PHYA‐E1, GI‐CO and microRNA‐dependent modules that are known to regulate flowering time in soybean. E1 is a soybean‐specific transcription factor, which is significantly suppressed under SD conditions but is induced under LD conditions. The expression of E1 is controlled by two phytochromeA (PHYA) homologs, E3 (Glyma19g41210) (Watanabe et al., 2009) and E4 (Glyma20g22160) (Liu et al., 2008), and exhibits a bimodal pattern at dawn and dusk under LD conditions (Xia et al., 2012). J, as the ortholog of Arabidopsis thaliana EARLYFLOWERING3 (ELF3) (Yue et al., 2017), acts upstream of E1 in the soybean flowering pathway and down‐regulates E1 transcription to relieve repression of GmFT2a and GmFT5a under SD conditions. In addition, expression of J is suppressed by the combined action of E3 and E4 under SD conditions (Lu et al., 2017).

GmFT2a and GmFT5a are up‐regulated under SD conditions, whereas they are significantly down‐regulated under LD conditions (Kong et al., 2010; Xu et al., 2013). Interestingly, a previous SD‐to‐LD transfer experiments showed that GmFT2a expression was more sensitive to photoperiodic changes than that of GmFT5a (Kong et al., 2010), and it is worth noting that only the GmFT5a transcripts accumulated in the late growth stage of soybean under LD conditions. These results indicated that, in addition to PHYA‐mediated flowering pathways, GmFT5a may be controlled in a photoperiod‐independent manner by another regulatory mechanism in longer day lengths (Kong et al., 2010). An ortholog of Arabidopsis thaliana GIGANTEA (GI), GmGIa (Glyma10g36600), is responsible for the E2 locus. Under natural day length conditions, E2 inhibits the expression of GmFT2a, whereas the expression level of GmFT5a is not controlled by the E2 locus (Watanabe et al., 2011).

Under LD conditions, the expression of GmmiR156b, a flowering suppressor in soybean, is up‐regulated by E1, E2, E3, and E4, and can suppress E1 (E1‐Like) and E2 (E2‐Like) genes. Overexpression of GmmiR156b caused a significantly late‐flowering phenotype under LD conditions, whereas it caused a slightly late flowering under SD conditions. Importantly, the expression level of GmFT5a is down‐regulated by the overexpression of GmmiR156b under LD or SD conditions, whereas GmFT2a is decreased only under SD conditions (Cao et al., 2015). Both GmFT2a and GmFT5a proteins interact with the bZIP transcription factor GmFDL19 in soybean and then up‐regulate several downstream flowering‐related genes (such as GmAP1, GmSOC1 and GmLFY) in both a redundant and differential pattern, suggesting that the FT/FD‐AP1 module is conserved in soybean (Nan et al., 2014). Overexpression of GmFT2a or GmFT5a in the soybean variety Williams 82 promotes early flowering under LD conditions (Nan et al., 2014).

The soybean E9 has been confirmed as GmFT2a, and the transcriptional expression level of its recessive allele e9 (E9 and e9 contain the same coding sequence) is always lower than that of E9 under any photoperiod conditions, and this results from a Ty1/copia‐like retrotransposon SORE‐1 that is highly methylated and inserted in the first intron of e9, thus cause late‐flowering phenotype (Zhao et al., 2016). In a previous study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a in the soybean variety Jack. Site‐directed mutations in the first exon of GmFT2a generated frameshift mutations and then damaged the gene functions. The homozygous ft2a mutants exhibited late‐flowering phenotype under both LD and SD conditions (Cai et al., 2018).

GmFT2a and GmFT5a often appear in pairs as flowering promoters in previous studies. However, information about the individual effects of GmFT2a and GmFT5a in floral induction and their differential regulation of downstream flowering‐related genes under SD and LD conditions remain limited. In this study, we created transgenic plants overexpressing GmFT2a or GmFT5a (GmFT2a‐ox plants, GmFT5a‐ox plants), ft2a mutants, ft5a mutants and ft2aft5a double mutants to dissect individual effects of GmFT2a and GmFT5a on flowering time under LD and SD conditions.

Results

Genetic effects of GmFT2a and GmFT5a on flowering time under different photoperiods

The soybean variety ZGDD showed extremely late flowering under LD conditions, but not under SD conditions, whereas variety HH27 showed early flowering under LD and SD conditions. The 308 F6:7 RILs (recombination inbred lines) developed from crosses between ZGDD and HH27 as the parental lines were planted with two replicates in randomized complete blocks in Beijing (N40°13′,E116°33′) on 3 July 2016 and 17 June 2017, Xinxiang (N35°08′, E113°45′) on 5 July 2016 and 22 June 2017, Sanya (N18°18′, E109°30′) on 19 December 2016 and 18 March 2017, Jining (N35°27′, E116°34′) on 3 July 2016 and Xiangtan (N27°40′, E112°39′) on 20 June 2017, and these eight environments were named 16BJ, 17BJ, 16XX, 17XX, 16SY, 17SY, 16JN and 17XT, respectively (Figure 1a). The 308 RIL population was genotyped, and a high‐density genetic map with a total length of 2208.16 cm, comprising 20 linkage groups with an average interval between adjacent markers of 0.64 cm, was constructed.

Figure 1.

Figure 1

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Genetic effects of qFT16‐1 and qFT16‐2 under different photoperiods. The eight environments: Beijing on 3 July 2016 and 17 June 2017, Xinxiang on 5 July 2016 and 22 June 2017, Sanya on 19 December 2016 and 18 March 2017, Jining on 3 July 2016, and Xiangtan on 20 June 2017, were named 16BJ, 17BJ, 16XX, 17XX, 16SY, 17SY, 16JN and 17XT, respectively. (a) Geographic distribution of these environments in China. (b) The dotted line represents the LOD (likelihood of odd) score = 3.2. (c) PVE of qFT16‐1 and qFT16‐2 in the eight environments. (d) Additive effects of qFT16‐1 and qFT16‐2 in the eight environments.

Based on the genetic map, two major effect QTLs for flowering time—qFT16‐1 and qFT16‐2, both on chromosome 16—were identified across all eight environments (Figure 1b and Table S1). The major QTL qFT16‐1 explained 2.9%–20.07% of the phenotypic variance. The other major QTL, qFT16‐2, accounted for 1.88%–18.66% of the phenotypic variation. To validate our QTL mapping results, we compared the stable QTL qFT16‐1 and qFT16‐2 with the previously reported QTLs and genes involved in soybean flowering time regulation; two major previously reported flowering accelerator (Kong et al., 2010; Takeshima et al., 2016; Zhao et al., 2016) genes (GmFT5a and GmFT2a) are located, respectively, in the genomic regions wherein we detected qFT16‐1 and qFT16‐2. We found no frameshifts or premature stop codons in the coding regions of GmFT2a or GmFT5a in HH27 and ZGDD (Appendix S1). Although the GmFT5a coding regions were identical, Takeshima et al. (2016) detected 15 DNA polymorphisms between parents with the early‐flowering and late‐flowering alleles in the promoter region, an intron, and the 3′ untranslated region of GmFT5a, and confirmed that GmFT5a is the gene responsible for qDTF‐J1. Sun et al. (2011) demonstrated that the expression of GmFT2a in HH27 is higher than that in ZGDD under LD and SD photoperiods. Thus, GmFT5a and GmFT2a are the most likely candidate genes underlying the qFT16‐1 and qFT16‐2 QTLs we identified. Interestingly, compared with qFT16‐2, the additive effect and the PVE value of qFT16‐1 was much larger under the six environments with relatively longer day lengths (16BJ, 17BJ, 16XX, 17XX, 16JN and 17XT) but were substantially lower for the two environments with SD conditions (16SY and 17SY) (Figure 1c,d and Figure S1). These results indicate that GmFT5a and GmFT2a apparently exert distinct genetic effects under different photoperiods.

GmFT2a and GmFT5a have different effects on flowering time under SD and LD conditions

Combining the aforementioned results, we suggest that GmFT5a and GmFT2a have different genetic effects on flowering time under different photoperiods: GmFT5a has more effects than GmFT2a in higher latitudes. To further evaluate this hypothesis, we examined the flowering times of WT plants, GmFT2a‐ox plants, GmFT5a‐ox plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants. Under SD (12 h light/12 h dark) conditions and compared to wild‐type (WT) plants, the GmFT2a‐ox plants displayed early flowering by about 9 days and the ft2a mutants exhibited late flowering by about 6 days (16.7 ± 1.5 DAE for GmFT2a‐ox plants vs. 32.0 ± 1.3 DAE for ft2a mutants vs. 26.1 ± 1.7 DAE for WT) (Figure 2a,e,g). The flowering time of GmFT5a‐ox plants and ft5a mutants was almost the same as WT plants (25.6 ± 1.2 DAE for GmFT5a‐ox plants vs. 26.9 ± 1.4 DAE for ft5a mutants vs. 26.1 ± 1.7 DAE for WT) (Figure 2c,e,g). The ft2aft5a double mutants flowered at 57.4 ± 3.5 DAE, about 31 days later than WT plants (Figure 2e,g).

Figure 2.

Figure 2

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(a) Flowering time of WT plants and GmFT2a‐ox plants under SD conditions. (b) Flowering time of WT plants and GmFT2a‐ox plants under LD conditions. (c) Flowering time of WT plants and GmFT5a‐ox plants under SD conditions. (d) Flowering time of WT plants and GmFT5a‐ox plants under LD conditions. (e) Flowering time of WT plants, ft2a mutants, ft5a mutants, and ft2aft5a double mutants under SD conditions. (f) Flowering time of WT plants, ft2a mutants, ft5a mutants, and ft2aft5a double mutants under LD conditions.

Under LD (16 h light/8 h dark) conditions and compared to WT plants, the GmFT2a‐ox plants showed extremely early‐flowering phenotypes of about 32 days, while the ft2a mutants exhibited late flowering by about 3.6 days (16.8 ± 1.2 DAE for GmFT2a‐ox plants vs. 52.1 ± 1.5 DAE for ft2a mutants vs. 48.5 ± 1.4 DAE for WT) (Figure 2b,f,g). The flowering times of the GmFT2a‐ox plants were basically same between SD and LD conditions, suggesting that the overexpression of GmFT2a promotes precocious flowering independent of the photoperiod. Unlike the observation of no significant phenotypic alterations in flowering time that we observed under SD conditions, we found that overexpression of GmFT5a caused early flowering by about 16 days (32.2 ± 1.9 DAE for GmFT5a‐ox plants vs. 48.5 ± 1.4 DAE for WT) under LD conditions (Figure 2d,g). Even more remarkably, the ft5a mutants flowered at 124.7 ± 15.4 DAE, about 76 days later than WT plants (Figure 2f,g). The ft2aft5a double mutants did not flower up to 150 DAE (Figure 2g).

Synchronously, as shown in Figure S2, under SD13‐LD conditions (shifted to LD after 13 days of SD treatment), the flowering time of WT plants was 30.2 ± 1.0 DAE, about 4 days later than under SD and 18 days earlier than under LD. These results indicate that the activation of flowering as induced by SD conditions can promote flowering even under non‐inductive conditions (LD). The ft2a mutants flowered at 37.2 ± 2.0 DAE, exhibiting late flowering by about 7 days compared to WT plants (SD13‐LD). And, more remarkably, the ft5a mutants (SD13‐LD) had the same extreme late‐flowering phenotype (126.4 ± 13.5 DAE) as they did under LD conditions (124.7 ± 15.4 DAE). That is to say, the activation of flowering as induced by GmFT2a under SD conditions could not completely make up for the inactivation of GmFT5a gene under LD conditions. Together, we conclude that GmFT2a and GmFT5a collectively regulate flowering time in soybean, but the effect of GmFT2a is apparently more prominent than that of GmFT5a under SD conditions. Conversely, the effect of GmFT5a is apparently more prominent than that of GmFT2a under LD conditions. Thus, the flowering regulation pathways which include GmFT2a and GmFT5a as participants are different under SD and LD conditions.

The effect of GmFT5a is dependent on day length in flowering time regulation

Because the ft5a mutants exhibited extreme late‐flowering phenotype under LD conditions, but flowered similar to the WT plants under SD conditions, we assumed that the different effects of GmFT5a on flowering time regulation may require a critical photoperiod to transform. To test the idea, the WT plants, ft2a mutants and ft5a mutants were grown under 14H (14 h light/10 h dark) and 15H (15 h light/9 h dark) photoperiodic conditions. As shown in Figure S3, under 14H conditions, the ft2a mutants exhibited late flowering by about 3.6 days compared to WT plants (33.6 ± 1.2 DAE for ft2a mutants vs. 30.0 ± 0.5 DAE for WT), and the flowering time of ft5a mutants was a slightly but significantly later than that of WT plants (31.1 ± 0.5 DAE for ft5a mutants vs. 30.0 ± 0.5 DAE for WT). Under 15H conditions, the ft2a mutants also exhibited late flowering by about 3.7 days compared to WT plants (34.9 ± 0.8 DAE for ft2a mutants vs. 31.2 ± 1.2 DAE for WT), and the ft5a mutants exhibited late‐flowering phenotype by about 6.8 days compared to WT plants (38.0 ± 1.6 DAE for ft5a mutants vs. 31.2 ± 1.2 DAE for WT).

We summarized the data of flowering time under SD, 14H, 15H and LD conditions and found that the flowering time of WT plants was delayed gradually as day length extended from 12 h to 15 h, but was then delayed significantly under 16 h light/8 h dark conditions. We therefore suggest that the critical photoperiod of soybean variety Jack occurs between 15 h and 16 h. As the day length gradually increased, the degree of the late‐flowering phenotype of the ft5a mutants became more and more significant. Intriguingly, the ft5a mutants exhibited extreme late‐flowering phenotype under LD conditions, about 76 days later than that of WT plants. These results indicated that the flowering regulation pathways in soybean changed with photoperiod from SD to LD conditions. There is apparently an important flowering regulation pathway that enables short‐day plant soybean adapt to LD conditions, and GmFT5a but not GmFT2a is apparently essential for this pathway.

The ft2aft5a double mutants showed late flowering and produced more pods and seeds under SD conditions

Under SD conditions, the maturity R7 stage (Fehr et al., 1971) of the WT plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants was 77.2 DAE, 80.4 DAE, 76.1 DAE and 97.7 DAE, respectively (Figure 3a). The WT plants produced an average of two additional nodes after floral induction and terminated the stem growth prematurely, whereas the ft2a mutants exhibited late flowering by about 6 days and maintained a relatively long period of vegetative growth after flowering, ultimately resulting in higher heights and the growth of an average of four additional as compared to the WT plants. In contrast, the plant height and node number of the ft5a mutants were not significantly different from WT plants (Figure 3b,c,d). It is highly notable that the vegetative growth of the ft2aft5a double mutants under SD conditions was very vigorous, and they flowered at 57.4 ± 3.5 DAE, about 31.3 days later than that of WT plants. The number of nodes per ft2aft5a double mutant was 20.0 ± 0.9, while that of WT plants was only 10.3 ± 0.7 (Figure 3b,c). These mutants reached a height of 235 cm, over double the height of WT plants (Figure 3b,d).

Figure 3.

Figure 3

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Growth of the WT plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants under SD (12 h light/12 h dark) conditions. (a) Maturity (R7 stage) is shown as the mean values ± standard deviation. DAE, days after emergence. (b) Scale bars, 20 cm. (c) and (d) Number of nodes and plant height are shown as the mean values ± one standard deviation. (e) Flower stalks of WT plants, ft2a mutants and ft5a mutants located directly at the leaf axil. In contrast, most nodes of the ft2aft5a double mutants first produced many new branches instead of flowers at the leaf axil and then produced flower buds at these new branches. (f) New branches at the leaf axil of the ft2aft5a double mutants produced more pods and seeds. (g) and (h) Number of pods and seeds are shown as the mean values ± one standard deviation.

In general, the flowering position of WT plants was mainly at the leaf axil; flower stalks are located at this position. The ft2a mutants and ft5a mutants had the same flowering position as the WT plants, whereas most nodes of the ft2aft5a double mutants first produced many new branches instead of flowers at the leaf axil and then produced flower buds at these new branches (Figure 3e). Importantly, the branches at the leaf axils of the ft2aft5a double mutants produced more pods than the WT, thereby increasing the number of pods at each node (Figure 3f). Along with the increased number of nodes, the total numbers of pods and seeds of per ft2aft5a double mutant (pods 54.7 ± 12.6; seeds 106.3 ± 22.8) were significantly more than that of WT plants (pods 11.9 ± 3.2; seeds 27.7 ± 6.4) (Figure 3g,h).

Expression of flowering‐related genes in the shoot apex under SD and LD conditions

To explore how GmFT2a and GmFT5a regulate flowering differently under SD and LD conditions, we focused on several downstream flowering‐related genes which have been isolated and characterized in soybean (Chi et al., 2011; Jia et al., 2015; Meng et al., 2007; Na et al., 2013; Nan et al., 2014; Zhong et al., 2012). We analysed the expression level of such genes, including GmAP1a (Glyma16g13070), GmAP1b (Glyma01g08150), GmAP1c (Glyma08g36380), GmLFY2 (Glyma06g17170), GmFULa (Glyma06g22650), GmFULb (Glyma04g31847), GmAG (Glyma15g09500), GmSOC1a (Glyma18g45780) and GmSOC1b (Glyma09g40230), in shoot apices. GmFT2a and GmFT5a expression were significantly up‐regulated, respectively, in GmFT2a‐ox and GmFT5a‐ox plants (Figures S4 and S5). The expression levels of GmAP1 (a, b, c), GmLFY2, GmFULa, GmFULb and GmAG were significantly up‐regulated in GmFT2a‐ox plants (Figure S4), findings consistent with the extreme early‐flowering phenotype observed for these plants under both SD and LD conditions. Under SD conditions, the expression levels of GmAP1 (a, b, c) and GmAG were not affected, while those of GmLFY2, GmFULa and GmFULb were slightly but not significantly up‐regulated in GmFT5a‐ox plants. In contrast, under LD conditions, GmAP1 (a, b, c) and GmAG expression were significantly up‐regulated in GmFT5a‐ox plants (Figure S5), which may contribute to its early‐flowering phenotype.

As shown in Figure 4, GmFT2a and GmFT5a expression were significantly down‐regulated in ft2a mutants and ft5a mutants, respectively. They were also significantly down‐regulated in ft2aft5a double mutants. Under SD conditions, GmAP1 (a, b, c) and GmAG were significantly down‐regulated in ft2a mutants and ft5a mutants, with particularly pronounced down‐regulation in the ft2aft5a double mutants. The expression of neither GmFULa nor GmFULb was significantly affected in the ft2a mutants or in the ft5a mutants, but they were significantly down‐regulated in the ft2aft5a double mutants. Viewed in combination with the flowering phenotype results mentioned above, we suggest that GmFT2a and GmFT5a may act as the primary activators of flowering, even when either of them is non‐functional. GmFT2a has more significant effects on floral transition than that of GmFT5a under SD conditions. GmLFY2 expression was slightly down‐regulated in ft5a mutants and ft2aft5a double mutants, but not in ft2a mutants. Under LD conditions, GmAP1 (a, b, c), GmLFY2, GmAG, GmFULa and GmFULb were not affected in the ft2a mutants, whereas the expression of these genes was significantly down‐regulated in ft5a mutants and ft2aft5a double mutants, providing further evidence that GmFT5a is a crucial gene for soybean flowering under LD conditions. We did not find any significant expression change in GmSOC1a or GmSOC1b in the aforementioned plants under SD or LD conditions (Figure 4, Figures S4 and S5).

Figure 4.

Figure 4

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Expression analyses of GmFT2a, GmFT5a and flowering‐related genes in the shoot apices of the WT plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants. (a) Expression analyses under SD (12 h light/12 h dark) conditions. (b) Expression analyses under LD (16 h light/8 h dark) conditions. Relative transcript levels of these genes were normalized to GmActin. Average and SE (standard error) values for three replications are shown in these bar plots. **P < 0.01.

Haplotyping and analysis of phenotypic variation under natural field conditions in five different latitudes

We subsequently examined the nucleotide polymorphisms in the coding and non‐coding regions of the GmFT2a and GmFT5a loci in a diversity panel comprising 202 soybean accessions with varied flowering time. No frameshifts or premature stop codons were found in the coding regions of GmFT2a or GmFT5a. We also found that there was clear linkage disequilibrium for the GmFT2a or GmFT5a loci in this diversity panel (Figure S6). For GmFT2a, seven haplotypes were identified, and four major haplotypes (FT2a‐Hap1, FT2a‐Hap2, FT2a‐Hap3 and FT2a‐Hap4) with higher frequencies in the panel were further analysed (Figure 5a). For GmFT5a, thirteen haplotypes were identified, and four major haplotypes (FT5a‐Hap2, FT5a‐Hap3, FT5a‐Hap5 and FT5a‐Hap7) with higher frequencies in the panel were analysed in greater detail (Figure 5b).

Figure 5.

Figure 5

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Haplotypes of GmFT2a and GmFT5a. GmFT2a and GmFT5a gene regions of 202 soybean accessions were compared with those of Williams 82. Site numbering and physical positions are also based on the reference genome sequence of Williams 82. (a) Haplotypes of GmFT2a. (b) Haplotypes of GmFT5a. Nucleotides are highlighted in different colours. The number of cultivars belong to each haplotype is listed in the right column.

The flowering time phenotypes were also recorded for plants grown at five sites in China with different latitudes: Sanya (N18°21′, E109°10′), Hunan (N27°49′, E112°56′), Beijing (N40°09′, E116°14′), Changchun (N43°49′, E125°21′) and Heihe (N50°15′, E127°27′). Consistent with its most northerly position, the soybean accessions grown at Heihe showed the longest flowering time of the five environments. Furthermore, by comparing the flowering times of the different GmFT2a or GmFT5a haplotypes (Figure S7a) and these combined haplotypes (Figure S7b), we found that haplotype FT5a‐Hap2 showed earlier flowering time in the five environments, and the phenotypic tendency becomes more pronounced with the increasing latitude. We also found that the haplotype FT5a‐Hap3 played an important role in ‘extremely’ late flowering, especially at higher latitudes (Figure S7a). The combined haplotype FT2a‐Hap2/FT5a‐Hap2 exhibited significant early flowering in all five environments. All varieties with the FT2a‐Hap3/FT5a‐Hap3 haplotypes could not flower normally when they were grown at Heihe (higher latitude) (Figure S7b). In short, we conclude that different GmFT2a and GmFT5a haplotypes have considerable effects on the diversity of flowering time in soybean at different latitudes.

Distributions of major GmFT2a/GmFT5a haplotypes in soybean varieties from diverse geographical origins and maturity groups

Six ecological regions or ten subareas of soybean have been delineated in China on the basis of the climatic and geographical conditions, cropping systems, season sowing types and maturity group (MG) types (Wang and Gai, 2002). We analysed the geographic distribution of varieties with major GmFT2a/GmFT5a haplotypes. FT2a‐Hap1 was mostly found in the Huanghuaihai double cropping planting eco‐region (Figure S8a). FT2a‐Hap2 was mostly found in higher latitude region in the northern single cropping planting eco‐region and the Huanghuaihai double cropping planting eco‐region (Figure S8b). Accessions of FT2a‐Hap3 were present in the four eco‐regions located south of N32°18′ in China (Figure S8c). The geographic distribution of FT2a‐Hap4 was comparatively wide, but was rare in the northeast spring planting ecological subarea (Figure S8d).

Notably, FT5a‐Hap2 was only found in a higher latitude region: the northern single cropping planting eco‐region (Figure S8e). FT5a‐Hap3 was mostly found in the Huanghuaihai double cropping planting eco‐region and eco‐regions in the south of the Qinling Mountains‐Huaihe River, and it was also rare in the northeast spring planting ecological subarea (Figure S8f). We further analysed the maturity groups of accessions with major GmFT2a/GmFT5a haplotypes (Figure 6). The FT2a‐Hap1, FT2a‐Hap3, FT2a‐Hap4 and FT5a‐Hap3 genotypes were not found among the earlier maturing varieties (MG 000, MG 00 and MG 0). FT2a‐Hap2 was mainly distributed in the varieties of MG 1, MG 2 and MG 3. The distributions of FT5a‐Hap5 and FT5a‐Hap7 in various maturity groups were relatively dispersed. Most notably, FT5a‐Hap2 was only found in the earlier maturing varieties (from MG 000 to MG 3). These distributions were consistent with flowering responses to day length. We suggest that the FT5a‐Hap2 genotype may contribute to early flowering of soybean varieties at high latitudes.

Figure 6.

Figure 6

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Maturity groups of the soybean accessions with major haplotypes of GmFT2a and GmFT5a. The number next to coloured bar indicates the number of soybean accessions with the corresponding haplotype for GmFT2a or GmFT5a.

Discussion

GmFT2a and GmFT5a have been mentioned in a majority of studies that examined flowering time regulation in soybean, and they are often mentioned in pairs. There are 10 FT homologs in soybean (Kong et al., 2010). Among them, GmFT2a and GmFT5a have been identified as flowering activators and integrators (Kong et al., 2010; Nan et al., 2014; Sun et al., 2011), whereas GmFT1a (Glyma18g53680) and GmFT4 (Glyma08g47810) are known to be function as flowering repressors (Liu et al., 2018; Zhai et al., 2014). Previous studies have suggested that GmFT2a and GmFT5a can function redundantly in their roles as promoters of flowering, even though their expression levels respond differently to photoperiodic changes (Kong et al., 2010; Nan et al., 2014). However, information about the individual effects of GmFT2a and GmFT5a in floral induction and their differential regulation of downstream flowering‐related genes under SD and LD conditions remain limited. In this study, we demonstrated that the functions of GmFT2a and GmFT5a were not redundant as previously reported. GmFT2a and GmFT5a collectively regulate flowering time in soybean. Under SD conditions, the effect of GmFT2a was more prominent than that of GmFT5a, whereas GmFT5a had more significant effects than GmFT2a under LD conditions. GmFT5a, not GmFT2a, was essential for soybean to adapt to high latitude regions.

Soybean is sensitive to changes in day length. Although this photoperiod sensitivity limits its geographical range of cultivation, it has been widely introduced to many countries in various latitudes, such as Korea, Japan and in many countries of North/Central/South America. As a short‐day plant, a crucial trait for soybean to adapt to higher latitudes is a reduced or absent inhibition of flowering by LD conditions (Liu et al., 2016). In this study, we found four major haplotypes of GmFT5a. The relationship between flowering time and GmFT5a haplotype suggested that FT5a‐Hap2 is a functional allele contributing to early flowering at high latitudes, whereas FT5a‐Hap3 is a weakened allele contributing to ‘extremely’ late flowering. Thus, the haplotype data may be useful for flowering time prediction or make it easier to breed for varieties with the desired flowering time under LD conditions in future studies.

Many soybean varieties from mid and high latitudes that have been grown at lower latitudes flower and mature very early, which usually results in extremely low grain yields. Introduction of long‐juvenile traits could extend the vegetative phase and improve yield under SD conditions, thereby enabling expansion of cultivation in tropical regions (Lu et al., 2017). Moreover, the optimization of plant architecture and higher yield are also major goals of researchers. Consider that the plant architecture of soybean, including leaves, inflorescences, and pods at each node, is known to strongly influence soybean yields. Thus, to breed high‐yielding soybean varieties, the coordination between branching (branch numbers, lengths and angles) and vertical growth (main stem‐containing nodes) is required (Pedersen and Lauer, 2004). In a previous study, transgenic soybean plants overexpressing GmmiR156b produced significantly increased numbers of long branches, nodes, and pods, and they exhibited an increase in 100‐seed weight, achieving substantial improvements in soybean architecture and yield per plant (Sun et al., 2019). In the present study, we employed the CRISPR/Cas9 system to specifically knockout the soybean gene GmFT5a and also produced ft2aft5a double mutants. Under SD conditions, the ft2aft5a double mutants flowered at 57.4 ± 3.5 DAE, about 31 days later than WT plants, and they maintained a vertical growth habit. As the vegetative growth stage was extended, these mutants produced significantly increased numbers of nodes. Beyond that, most of their leaf axils produced new branches and then produced many more pods in these branches, resulting in substantial increase in numbers of pods and seeds per plant (Figure 3). In view of the adaptation to SD conditions and the increased yield per plant, we speculate that the ft2aft5a double mutants may have enormous potential to be introduced to the tropics.

In conclusion, we dissected individual effects of GmFT2a and GmFT5a on flowering time under LD and SD conditions, these results will contribute to more accurate studies on flowering regulation and expand the regional adaptability of fine soybean varieties in the future.

Experimental procedures

Genotyping and QTL analysis

In this study, the 308 RIL population was genotyped using a type IIB endonucleases restriction‐site associated DNA approach (2b‐RAD) (Wang et al., 2012), and a linkage map (2208.16 cm) with an average distance of 0.64 cm between adjacent markers was constructed using JoinMap 4.1 (Stam, 1993). QTL analysis for flowering time was performed according to the linkage map using QTL IciMapping software v4.1, with inclusive composite interval mapping of additive functionality (ICIM‐ADD) (Li et al., 2008a; Meng et al., 2015).

Soybean materials and growth conditions

The sequence and detailed information of the soybean genes GmFT2a and GmFT5a was searched from the Phytozome website (phytozome.jgi.doe.gov). To construct overexpression vectors of GmFT2a and GmFT5a, the total RNA from fully developed trifoliate leaves of the soybean cultivar Jack (grown under SD conditions) was isolated using a TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) and single‐stranded cDNA wad synthesized using TransScript One‐Step gDNA Removal and cDNA Synthesis Super Mix (TransGen Biotech, Beijing, China). The CDS of GmFT2a (531 bp) and GmFT5a (519 bp) were, respectively, amplified by primers GmFT2a‐ox‐F/R and GmFT5a‐ox‐F/R (Table S2) and then inserted into the plant binary vector pTF101.1 using a pEASY‐Uni Seamless Cloning and Assembly Kit (TransGen Biotech, Beijing, China). GmFT2a and GmFT5a were driven by a 2X CaMV 35S promoter. Each construct was transformed into Agrobacterium tumefaciens strain EHA101 via electroporation. The variety Jack was used for transformation according to a previously published protocol (Chen et al., 2018). Three transgenic GmFT2a T3 overexpression lines (#2, #4, #7) and three GmFT5a T3 overexpression lines (#1, #3, #4) were used for expression analysis of flowering‐related genes. The expression levels of GmFT2a and GmFT5a were highest in the GmFT2a overexpression line #2 and GmFT5a overexpression line #3, respectively, so they were used to study the flowering phenotype. The homozygous ft2a mutants (1‐bp insertion at target site GmFT2a‐SP2, frameshift mutation) that we previously reported (Cai et al., 2018) were used in this study.

We employed the CRISPR/Cas9 system to specifically knockout the soybean gene GmFT5a. Two target sites (referred to here as GmFT5a‐SP1 and GmFT5a‐SP2) in the first exon of GmFT5a were selected (Figure S9a) using the web tool CRISPR‐P (Lei et al., 2014). Pairs of DNA oligonucleotides of the two sgRNAs were synthesized by TSINGKE (Beijing) and annealed to generate dimers, which were subsequently integrated into the CRISPR/Cas9 expression vector we previously reported (Cai et al., 2018). These vectors were then individually transformed into Agrobacterium tumefaciens strain EHA105 via electroporation. The soybean variety Jack was used for transformation according to a previously reported protocol (Chen et al., 2018). We totally detected 34 T1 homozygous ft5a mutants, and obtained two types of mutations at target site GmFT5a‐SP1 (Figure S9b,c) and five types of mutations at target site GmFT5a‐SP2 (Figure S9b,d). We then performed hybridization using T1 homozygous ft5a mutants (2‐bp insertion at target site GmFT5a‐SP2) as the male parent and ft2a mutants (1‐bp insertion) as the female parent to generate ft2aft5a double mutants.

In this study, the culture rooms under LD (16 h light/30 °C and 8 h dark/22 °C), 14H (14 h light/30 °C and 10 h dark/22 °C), 15H (15 h light/30 °C and 9 h dark/22 °C), SD (12 h light/30 °C and 12 h dark/22 °C) and SD13‐LD (transferred to LD after 13 days of SD treatment) photoperiodic conditions were used. The red‐to‐blue quantum (R:B) ratio of the light was 5.03, while the red‐to‐far‐red quantum (R:FR) ratio of the light was 3.26.

Phenotyping and statistical analysis

The flowering time of each soybean plant was recorded as days from emergence to the R1 stage (one flower at any node), and the physiological maturity was recorded as days from emergence to the R7 stage (any pod becomes to the mature colour) (Fehr et al., 1971). The plant height was measured from cotyledon node to stem tip. The cotyledon node was counted as the first node. We also counted the number of pods and seeds per plant. Statistical analyses were performed using Microsoft Excel. A one‐way analysis of variance least significant difference test (LSD) was used to compare the significance of differences between controls and treatments at the 0.01 probability level. SigmaPlot 10.0 was used for drawing bar plots. These data are shown as the mean values ± one standard deviation.

Gene expression analysis by quantitative real‐time PCR

To examine the expression of flowering‐related genes in shoot apex, the shoot apices of the WT plants, GmFT2a‐ox plants, GmFT5a‐ox plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants under SD conditions were sampled at 13 DAE. The WT plants and GmFT2a‐ox plants under LD conditions were sampled at 13 DAE. The WT plants, GmFT5a‐ox plants, ft2a mutants, ft5a mutants and ft2aft5a double mutants under LD conditions were sampled at 30 DAE. Total RNA was extracted using a Quick‐RNA MicroPrep Kit (Zymo Research, Beijing, China). First‐strand cDNA was synthesized from the total RNA using TransScript One‐Step gDNA Removal and cDNA Synthesis Super Mix (TransGen Biotech, Beijing, China). For qRT‐PCR, each 10 μL reaction contained 1 μL 1:5 diluted cDNA with 0.2 μL upstream and downstream primers (10 μm), 3.6 μL ddH2O and 5 μL ChamQ SYBR® qPCR Master Mix (Vazyme Biotech, Nanjing, China). The ABI QuantStudio™ 7 Flex Real‐Time PCR System was used. The PCR cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and a primer extension reaction at 60 °C for 30 s. All PCRs were run with three biological replicates each. The relative expression level was analysed using the 2−ΔΔCt method with the GmActin (Glyma18g52780) gene as an internal control. Primers used in the expression analyses are listed in Table S2.

Conflict of interest

The authors declare that they have no conflicts of interest.

Author contributions

Y.C. and L.W. performed the experiments. Y.C., L.W. and L.C. wrote the manuscript. T.W. and L.L. provided the data for the loci in the 202 soybean accessions of the diversity panel. W.Y. assisted in soybean transformation. S.S. and C.W. provided soybean varieties. S.S. constructed the RIL population. S.Y. and B.J. revised the manuscript. W.H. and T.H. designed and advised on the experiments and revised the manuscript.

Supporting information

Figure S1 Day length of the eight environments.

Figure S2 Flowering time under SD13‐LD (shifted to LD after 13 d of SD treatment) conditions.

Figure S3 Flowering time of WT (wild‐type) plants, ft2a mutants and ft5a mutants under SD, 14H, 15H and LD conditions.

Figure S4 Expression analyses of GmFT2a and flowering‐related genes in shoot apices of three transgenic GmFT2a overexpression lines #2, #4, #7.

Figure S5 Expression analyses of GmFT5a and flowering‐related genes in shoot apices of three transgenic GmFT5a overexpression lines #1, #3, #4.

Figure S6 Linkage disequilibrium analysis in the coding and non‐coding regions of GmFT2a and GmFT5a among 202 soybean accessions.

Figure S7 Flowering time of the soybean accessions with major haplotypes of GmFT2a, GmFT5a and combined haplotypes of GmFT2a/GmFT5a at five different latitudes.

Figure S8 Geographic distribution of soybean accessions with major haplotypes of GmFT2a and GmFT5a.

Figure S9 Homozygous targeted mutagenesis of GmFT5a induced by CRISPR/Cas9.

Table S1 Putative QTL for soybean flowering time in RIL families across eight environments on chromosome 16.

Table S2 Primer sequences used in the present study.

Appendix S1 Genome sequences of GmFT2a and GmFT5a in soybean variety HH27 and ZGDD.

PBI-18-298-s001.pdf (4.9MB, pdf)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31871644), the Major Science and Technology Projects of China (2016ZX08010‐004), the Ministry of Science and Technology of China (2016YFD0100504) and the CAAS (Chinese Academy of Agriculture Sciences) Agricultural Science and Technology Innovation Project.

Contributor Information

Tianfu Han, Email: hantianfu@caas.cn.

Wensheng Hou, Email: houwensheng@caas.cn.

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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 Day length of the eight environments.

Figure S2 Flowering time under SD13‐LD (shifted to LD after 13 d of SD treatment) conditions.

Figure S3 Flowering time of WT (wild‐type) plants, ft2a mutants and ft5a mutants under SD, 14H, 15H and LD conditions.

Figure S4 Expression analyses of GmFT2a and flowering‐related genes in shoot apices of three transgenic GmFT2a overexpression lines #2, #4, #7.

Figure S5 Expression analyses of GmFT5a and flowering‐related genes in shoot apices of three transgenic GmFT5a overexpression lines #1, #3, #4.

Figure S6 Linkage disequilibrium analysis in the coding and non‐coding regions of GmFT2a and GmFT5a among 202 soybean accessions.

Figure S7 Flowering time of the soybean accessions with major haplotypes of GmFT2a, GmFT5a and combined haplotypes of GmFT2a/GmFT5a at five different latitudes.

Figure S8 Geographic distribution of soybean accessions with major haplotypes of GmFT2a and GmFT5a.

Figure S9 Homozygous targeted mutagenesis of GmFT5a induced by CRISPR/Cas9.

Table S1 Putative QTL for soybean flowering time in RIL families across eight environments on chromosome 16.

Table S2 Primer sequences used in the present study.

Appendix S1 Genome sequences of GmFT2a and GmFT5a in soybean variety HH27 and ZGDD.

PBI-18-298-s001.pdf (4.9MB, pdf)

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