CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean
The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants.
In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean.
Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.
KeywordsCRISPR/Cas9 Plastochron length Plant architecture Soybean SPL
CRISPR-associated system 9
Clustered regularly interspaced short palindromic repeat
Cetyltrimethyl ammonium bromide
DENSE AND ERECT PANICLE 1
Flower Locus T
Glycine max INCREASED LEAF PETIOLE ANGLE 1
IDEAL PLANT ARCHITECTURE 1
IPA1 INTERACTING PROTEIN 1
Quantitative trait loci
Shoot apical meristem
SQUAMOSA PROMOTER BINDING PROTEIN
SQUAMOSA PROMOTER BINDING PROTEIN-LIKE
TEOSINTE BRANCHED 1
TEOSINTE GLUME ARCHITECTURE
WEALTHY FARMER’S PANICLE
Soybean (Glycine max) plant architecture is an important trait for developing high-yield cultivars, and this trait can be determined based on stem growth habit, node number, plant height, internode length, branch number, leaf size and shape [1, 2]. Previous studies on soybean plant architecture have primarily focused on stem growth habit [1, 3, 4, 5, 6, 7]. Recently, Gao et al. (2017) found that the Glycine max INCREASED LEAF PETIOLE ANGLE 1 (GmILPA1), a gene encoding an ‘anaphase-promoting complex/cyclosome’ (APC/C) protein, modulated the leaf petiole angle in soybean . In addition, marker-assisted studies have revealed many quantitative trait loci (QTLs) associated with various traits related to plant architecture in soybean, including plant height, internode length, node number, branch number, pod number, and leaflet length and width (http://www.SoyBase.org). However, the molecular mechanisms regulating plant architecture and yield potential remain unknown, and information about the genes responsible for improving soybean plant architecture is still limited.
In plants, most members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor (TF) family are regulated through miR156, and these TFs affect the transition between the juvenile and adult phases [9, 10, 11, 12]. In Arabidopsis, SPL9 and SPL15 have been shown to be implicated in the regulation of plastochron length and leaf size [13, 14]. In rice (Oryza sativa), OsSPL14 has been identified as IDEAL PLANT ARCHITECTURE 1 (IPA1) or WEALTHY FARMER’S PANICLE (WFP) gene, which regulates shoot branching during the vegetative phase and the number of grains produced in a panicle [15, 16]. The rice OsSPL14 gene encodes the closest homologous protein of the Arabidopsis SPL9 and SPL15, and its overexpression also prolongs plastochron length . The OsSPL14 can directly bind to the promoter of the TEOSINTE BRANCHED 1 (TB1) in rice to suppress rice tillering, and positively and directly regulates the expression of DENSE AND ERECT PANICLE 1 (DEP1) to affect plant height and panicle length . Wang et al. (2015) reported that a spatiotemporally coordinated gene network comprising the miR156/miR529/SPL and miR172/Apetala2 (AP2) pathways controls tiller and panicle branching in rice . Recently, Wang et al. (2017) identified a RING-finger E3 ligase, named IPA1 INTERACTING PROTEIN 1 (IPI1), that can interact with OsSPL14 in the nucleus . IPI1 promotes the degradation of OsSPL14 in panicles, while it stabilizes OsSPL14 in shoot apexes, thereby regulating plant architecture in rice . In soybean, transgenic plants overexpressing the GmmiR156b produced greatly altered plant architecture, leading to a remarkable increase in grain yield per plant . It has also been reported in soybean that the GmSPL9d gene is expressed in the shoot apical meristem (SAM) and axillary meristem (AM), and that GmSPL9d may regulate axillary bud formation and shoot branching by physically interacting with the homeobox protein WUSCHEL (WUS), a central regulator of AM formation . GmmiR156b regulates soybean plant architecture mainly through the direct cleavage of SPL genes . However, our knowledge on the functions of GmSPL9 genes in controlling plant architecture is still limited in soybean.
Recently, the emergence of clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) technology has brought new opportunities to the field of genetic manipulation in plants [22, 23, 24]. It has attracted large attention, and its application has dramatically expanded in genome editing of many crops, including rice [7, 23], wheat (Triticum aestivum) [25, 26, 27], maize (Zea mays) [28, 29], oilseed rape (Brassica napus) , barley (Hordeum vulgare) , cotton (Gossypium hirsutum) , tomato (Solanum lycopersicum)  and soybean [34, 35, 36]. Very recently, Cai et al. (2018) have reported the successful application of the CRISPR/Cas9 system in soybean in mutating the gene Flower Locus T (FT), which resulted in delayed flowering time of mutated plants under both short-day and long-day conditions, suggesting that gene knock-out mediated by the CRISPR/Cas9 system in soybean research is feasible . However, research in soybean using the CRISPR/Cas9 system is still rare, due to the fact that soybean transformation is still a great challenge for most research groups. Furthermore, most of the targets of the successful applications of the CRISPR/Cas9 system in gene editing in soybean were single gene [34, 35, 36]. Here, we report the CRISPR/Cas9-based multiple gene editing system to target four SPL9 genes in soybean. T4-generation soybean mutant plants carrying different combinations of mutations exhibited a number of altered characteristics in plant architecture. Our findings indicate that the CRISPR/Cas9 system is a promising tool to advance soybean breeding.
Target selection and construction of the CRISPR/Cas9 vector system for mutagenesis of four GmSPL genes in soybean
Targeted mutagenesis of four GmSPL9 genes in soybean
There is currently not much knowledge with respect to the functions of GmSPL9a, GmSPL9b and GmSPL9c on the regulation of plant architecture. However, there was a report about the function of GmSPL9d with regard to its regulatory function in plant architecture. Specifically, overexpression of the GmSPL9d gene suppressed the branch number in Arabidopsis transgenic plants . Thus, given the redundant functions of the GmSPL9 genes, to obtain profound evidence for their genetic involvement in regulating plant architecture, we were interested in identifying the higher-order mutants, especially those that contain mutation in the GmSPL9d gene (Fig. 3e; Additional file 2: Table S1). The seeds of ten T1-generation plants (four T1–10 plants and six T1–20 plants) were sown, and the DNAs of 120 independent T2 plants (12 independent T2 plants from each T1-generation plant) were obtained. We then mixed the DNAs of 12 independent T2 plants from each T1-generation plant as one pooled DNA template for PCR, resulting in 10 DNA pools. Sequence analysis showed that there were no edited mutations in the two target sites of GmSPL9d (GmSPL9d-SP3 and GmSPL9d-SP4) among the examined T2 plants. When we obtained the T3-generation seeds, we conducted similar experiments to identify spl9d mutants. The pooled DNAs of T3–10–1-3 (mixed 12 plants) and T3–10–1-6 (mixed 12 plants) had a new edited type in GmSPL9d (spl9d allele, Additional file 2: Table S1). Further sequence analysis showed a 1-bp deletion in the target site of GmSPL9d-SP3 in T3–10–1-3-2 (Fig. 3e; Additional file 2: Table S1), resulting in the spl9a (−/−)/spl9b-1 (−/−)/spl9d (+/−) mutant. In addition, we also obtained a ‘transgene-clean’ spl9b-1 (−/−) mutant line from the T2–10–1-1 line using the selectable marker gene Bar for selection (Additional file 2: Table S1; Additional file 5: Figure S3). The T2–10–1-1 line had 1-bp deletion in the target site of GmSPL9b-SP1, and its T3 and T4-generation plants were all ‘transgene-clean’ homozygous spl9b-1 mutants. After four generations of selection, we obtained the ‘transgene-clean’ homozygous spl9b-1 single and the spl9a/spl9b-1/spl9c/spl9d homozygous quadruple mutants, and some other mutants that were still Bar-positive like spl9a (−/−)/spl9b-1 (−/−), spl9a (−/−)/spl9b-1 (−/−)/spl9c (+/−), spl9a (−/−)/spl9b-2 (−/−)/spl9c (+/−) and spl9a (−/−)/spl9b-1 (−/−)/spl9c (+/−)/spl9d (+/−) from our genome editing experiment (Additional file 2: Table S1).
The examined four SPL9 genes regulate soybean plant architecture
The examined four SPL9 genes regulate plastochron length in soybean
The T4-generation spl9b-1 single homozygous mutant showed no difference in number of trifoliates compared with WT, whereas the other T4 higher-order mutant plants examined showed shorter plastochron lengths in soybean compared with the spl9b-1 single mutant and WT plants (Fig. 4f). In particular, 50 days after emergence (DAE), WT, GmmiR156b-OX, spl9b-1, spl9a (−/−)/spl9b-1 (−/−)/spl9c (+/−) and spl9a (−/−)/spl9b-2 (−/−)/spl9c (+/−) exhibited trifoliate leaves on the main stem in an average number of 18.2, 22.8, 18.7, 20.5 and 20.2, respectively, while the spl9a (−/−)/spl9b-1 (−/−)/spl9c (+/−)/spl9d (+/−) and spl9a (−/−)/spl9b-1 (−/−)/spl9c (−/−)/spl9d (−/−) mutant plants, when analyzed together, had the average number of trifoliate leaves of 21.5 (Fig. 4f). In addition, both the T2-generation spl9a (−/−)/spl9b-1 (−/−) double mutant and GmmiR156b-OX plants had shorter plastochron lengths compared with WT plants (Additional file 6: Figure S4). These data indicated that these four GmSPL9 genes are implicated in regulation of plastochron length in soybean, perhaps under the control of GmmiR156b.
GmSPL9b regulates expression of the four GmSPL9 genes
CRISPR/Cas9 genome editing efficiency in soybean - single construct for multiple mutations
In the current study, we explored the CRISPR/Cas9 system for mutagenesis of four GmSPL9 genes by using a single plasmid construct in an attempt to make loss-of-function soybean mutants to assess the functions of these genes in regulation of plant architecture (Figs. 1-2). We designed four target adaptors (SP1, SP2, SP3 and SP4) and found that only the SP1 adaptor targeting GmSPL9a and GmSPL9b genes produced heterozygous mutants in T0-generation plants (Fig. 1; Additional file 2: Table S1). However, the editing continued to work with the constructed CRISPR/Cas9 system in plants of later generations (Bar-positive), and new mutants were obtained at different target sites (Additional file 2: Table S1). Our data indicated that the genome editing efficiency was low in T0-generation of soybean, but relatively high in T1-generation plants (Additional file 2: Table S1). Similar results were reported in Arabidopsis, which showed that the editing efficiency and editing types induced by CRISPR/Cas9 were relatively low, and both uniform and chimeric mutations were occurred in the T1-generation [38, 39, 40]. It has been suggested that Arabidopsis transformation methods using vegetative tissues might produce relatively low editing efficiency . Thus, the low editing efficiency in the soybean T0-generation observed in this study might also be caused by our transformation method using cotyledonary node. Furthermore, we observed that the SP2 and SP4 adaptors designed for three target sites (GmSPL9a and GmSPL9b genes, and GmSPL9d gene, respectively) showed no edits in T0-, T1-, T2-, T3- and T4-generation plants. Previous studies reported that selection of target sequences with relatively higher GC content might result in a higher editing efficiency . We should select target sites with higher GC content to improve the editing efficiency. Therefore, at least in the case of soybean, a crop with low transformation frequency , less target genes with more target sites in each target gene should be designed to make sure that lower-order mutants can be generated. Subsequently, higher-order mutants can be obtained through crossing.
Functions of the four examined GmSPL9 genes in regulating plant architecture of soybean
In rice, many studies have reported that the OsSPL14 had a great role in regulation of plant architecture [15, 16, 17, 18, 19, 20]. In Arabidopsis, SPL9, SPL15 and SPL10 function in a redundant manner to regulate plastochron length [13, 14]. In bread wheat, miR156 was reported to control plant architecture via the repression of a group of SPL genes . The SPL TFs share a highly conserved DNA-binding domain called SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box . Several maize SBP-box-type TFs, such as TEOSINTE GLUME ARCHITECTURE (TGA1) , TASSELSHEATH4 (TSH4) , UNBRANCHED2 (Ub2) and UNBRANCHED3 (Ub3) , were shown to be associated with maize grain architecture. In soybean, a previous study reported that overexpression of GmmiR156b improved plant architecture, and consequently grain yield . Ectopic overexpression of GmSPL9d reduced branch number in Arabidopsis . However, the functions of GmSPL9d and its closest homologs, like GmSPL9a, GmSPL9b and GmSPL9c TFs (Additional file 1: Figure S1), in influencing soybean plant architecture remained to be determined.
Using the CRISPR/Cas9 system for genome editing, we were able to produce a number of single and higher-order mutants, particularly the quadruple mutant, for investigating the roles of these four TFs in forming soybean plant architecture. Specifically, we obtained the homozygous mutants spl9b-1 and spl9a/spl9b-1/spl9c/spl9d after 4 generations (Additional file 2: Table S1). Detailed analyses of the mutants in different generations showed that the T4 higher-order mutants carrying various combinations of mutations exhibited various increased levels in node number on main stem, total node number per plant, branch number and dry weight compared with WT and spl9b single mutant plants (Fig. 4). Our results suggest that these four GmSPL9 TFs might very likely regulate these characteristics of plant architecture in soybean. All lower- and higher-order mutants in all combinations of these four GmSPL9 genes should be obtained to clearly classify the important level of each of these four TFs in formation of soybean plant architecture. A new genome editing design is required to fulfill this task.
As discussed previously, we just obtained one spl9b single mutant plants with Bar negative, but did not obtained other three single mutants with individual mutation in GmSPL9a, GmSPL9c and GmSPL9d genes in order to clearly classify the functional roles of these four TFs. Thus, we could use only this stable spl9b single mutant as an example to analyze the expression patterns of all four GmSPL9 genes. Our results showed that the spl9b single mutant plants had higher expression levels of all four GmSPL9 genes in both SAM and leaves than WT (Fig. 5), while the transgenic plants overexpressing GmSPL9b displayed lower expression levels of GmSPL9a, GmSPL9c and GmSPL9d genes than WT plants (Additional file 7: Figure S5). This finding suggested that the GmSPL9b TF might negatively regulate the expression of GmSPL9 genes in soybean, which might result in no or minor changes in the spl9b single mutant, when compared with WT plants, with respect to the examined phenotypic parameters (Fig. 4). Further studies need to be conducted using all combinations of single, double, triple and quadruple mutant plants of these four GmSPL9 genes to examine their complex functions in soybean plant architecture and the feedback mechanism underlying the expression patterns of the examined GmSPL9 genes. In addition, our data were obtained under artificial climate chamber conditions. Further studies under field conditions are required to reveal the roles of these four GmSPL9 genes in regulating soybean plant architecture and especially grain yield, prior to using them in genetic engineering for improvement of soybean productivity.
The CRISPR/Cas9 system currently has become a versatile tool to advance crop plant breeding. In our present study, we used CRISPR/Cas9-based multiple genome editing, and successfully obtained several mutants, including the quadruple mutant, for assessment of the functions of four closely homologous GmSPL9 genes in formation of soybean plant architecture. Using these mutants, we found that the four GmSPL9 genes may have redundant or independent roles in regulating soybean plant architecture, depending on the phenotypic trait(s) examined. Our data also suggested that the GmSPL9b gene can regulate the expression of the four GmSPL9 genes, including itself, in soybean. Taken together, results of our studies improve the understanding of the application of CRISPR/Cas9 system and provide more knowledge on the regulation of plant architecture in soybean.
Plant materials and growth conditions
The soybean [Glycine max (L.) Merr.] cultivar Williams 82 was used for transformation. The WT plants, GmmiR156b-overexpressing transgenic plants (line #5 from Sun et al., 2018) , and mutant plants were cultivated in an artificial climate chamber under the conditions of 12-h light and 12-h dark photoperiod at 24 °C.
To investigate the plant architecture of transgenic plants, 17 T4-generation higher-order spl9 mutant plants of different combinations (Fig. 4) were grown in an artificial climate chamber under the 15-h light/9-h dark photoperiodic conditions at 28 °C. The WT, GmmiR156b-overexpressing transgenic and the homozygous spl9b-1 single mutant (Bar negative) plants were grown with 6 seedlings/each genotype (Fig. 4). The seeds of each genotype were germinated on moistened filter paper for 4 days at 28 °C and 60% humidity under 15-h light/9-h dark photoperiodic conditions. Germinated seedlings were transferred into 25 cm × 25 cm pot with each pot containing one seedling. All examined phenotypic parameters, including branch number (first branch number on main stem), node number on main stem and total node number per plant, were recorded at 50 DAE. To investigate the plastochron length in the T4 soybean mutants, the trifoliate leaves on main stem were recorded every 10 DAE from 20 to 50 DAE.
Construction of phylogenetic tree
The full-length protein sequences of 43 soybean SPLs were retrieved from the Phytozome (www. Phytozome.net/) and used to construct a phylogenetic tree to study the relationships of the soybean SPLs with the AtSPL9, AtSPL15, OsSPL14 and OsSPL17 proteins, whose full-length protein sequences were also downloaded from Phytozome. MEGA v.7.0 was used to construct the Neighbour-Joining unrooted tree .
Plasmid construction and soybean transformation
The nucleotide sequence of the four GmSPL genes were downloaded from Phytozome. The target sequence adaptors were designed using the web tool CRISPR-P (http://cbi.hzau.edu.cn/crispr/). The kanamycin resistance gene in the pYLCRISPR/Cas9P35S-B, which was received from Ma et al. , was replaced by the spectinomycin resistance aadA gene, resulting in the pYLCRISPR/Cas9P35S-BS. The four target sequence adaptors were integrated into different sgRNA expression cassettes and built into the pYLCRISPR/Cas9P35S-BS vector according to the protocol reported by Ma et al. (2015) . Briefly, a digestion/ligation reaction for each sgRNA expression cassette was prepared as follows: 1 μL 10 × CutSmart buffer, 20 ng pYLsgRNA plasmid DNA, 0.5 μL target adapter, 3 U BsaI-HF, 20 U T4 DNA ligase, 0.5 μL 10 × NEB T4 DNA ligase buffer and deionized H2O to a final volume of 10 μL. Subsequently, the digestion/ligation reaction was performed in a thermal cycler without using a heated lid at the following thermal cycling program: 10 cycles of 5 min at 37 °C and 5 min at 20 °C. Next, all four sgRNA cassettes were amplified by PCR using the products of digestion/ligation reaction as template and the site-specific primer pairs (Additional file 8: Table S2) at the following thermal cycling program: 22 cycles of 10 s at 95 °C, 15 s at 58 °C and 20 s at 68 °C. Equal amounts of the obtained PCR products were mixed and purified using a PCR product purification kit (Axygen, California, USA). Finally, a digestion/ligation reaction was prepared to assemble the four sgRNA cassettes into the pYLCRISPR/Cas9P35S-BS as follows: 1.5 μL 10 × CutSmart buffer, 100 ng pYLCRISPR/Cas9P35S-BS plasmid DNA, 100 ng pooled sgRNA cassettes (the mixture of four PCR products obtained from the previous step), 10 U BsaI-HF, 40 U T4 DNA ligase, 0.5 μL 10 × NEB T4 DNA ligase buffer and deionized H2O to a final volume of 15 μL. Subsequently, the digestion/ligation reaction was performed in a thermal cycler without using a heated lid at the following thermal cycling program: 15 cycles of 5 min at 37 °C, 5 min at 10 °C and 5 min at 20 °C.
The obtained CRISPR/Cas9 plasmid carrying the sgRNA cassettes was transformed into A. tumefaciens strain EHA105, followed by the soybean transformation that was conducted according to the description previously reported by Cao et al., 2015  with some modifications. Briefly, sterilized Williams 82 seeds were germinated in B5 medium for one day, and then the one-day-old germinated seedlings were vertically cut at cotyledonary node, and any remaining axial shoot/bud parts attached to the cotyledonary node were removed. Subsequently, the explants were wounded with a scalpel and dipped into the A. tumefaciens strain EHA105 carrying the CRISPR/Cas9 plasmid with the sgRNA cassettes built-in. After 4 days of co-cultivation in co-cultivation medium, the explants were transferred into the shoot induction medium without glufosinate. Seven days later, the explants were transferred into the shoot induction medium with 10 mg L− 1 glufosinate for 2 weeks. Subsequently, the explants were cultured in the shoot elongation medium containing 5 mg L− 1 glufosinate. When the elongated shoots were about 3 cm, they were transferred to rooting medium without further selection. Glufosinate (160 mg L− 1) was applied until the first trifoliate appeared to screen for T0, T1 and T2 transformants.
To obtain transgenic soybean plants overexpressing GmSPL9b, the GmSPL9b-pTF101 vector harboring the GmSPL9b gene under the 35S promoter from cauliflower mosaic virus was used to transform the soybean cultivar Dongnong 50, according to the description previously reported by Cao et al., 2015 .
DNA extraction and mutation screening
Genomic DNA was extracted from the leaves of each independent T0, T1, T2, T3 and T4 plant and used for PCR. The target site sequences were amplified by PCR with sequence-specific primer sets (Additional file 8: Table S2), and the PCR products were then separated by electrophoresis on 1.0% agarose in 1 × TAE buffer. The purified DNA fragments were sequenced and analyzed. The successfully edited types could be identified via sequence peaks and alignment to the reference sequences. The heterozygous mutants showed overlapping peaks near the target site, and the homozygous mutants were identified by sequence alignment with the WT sequence.
To screen the spl9d mutants in T2 generation, the seeds obtained from 10 T1-generation plants (four T1–10 plants and six T1–20 plants) were sown to sample DNAs from 120 independent T2 plants (12 independent T2 plants from each T1-generation plant). The DNAs of 12 independent T2 plants from each T1-generation plant were then pooled as a DNA template for PCR, resulting in a total of 10 DNA pools. To screen the spl9d mutants in T3 generation, similar protocol was conducted as described above in T2 generation. To screen the spl9d mutants in T4 generation, the DNA of every independent T4 plant was used as template for PCR. The PCR products were digested with EcoRI (New England Biolabs) and then separated by 1.0% agarose in 1 × TAE buffer.
RNA isolation, cDNA synthesis and qRT-PCR analysis
The shoot apical meristem (SAM) and third fully developed trifoliate leaves from the bottom of the plants were sampled at 20th DAE and immediately frozen in liquid nitrogen. Total RNA was isolated from each sample using TRIzol reagent (Invitrogen, USA), and cDNA was synthesized for quantitative RT-PCR (qRT-PCR) to assess the transcript levels of GmSPL9a (Glyma.02G177500), GmSPL9b (Glyma.09G113800), GmSPL9c (Glyma.03G143100), GmSPL9d (Glyma.19G146000) and GmTUB (Glyma.08G014200) (as an internal control) as described previously in Cao et al., (2015) . The primers used for qRT-PCR are listed in Additional file 8: Table S2. The qRT-PCR mixture was prepared by mixing 1 μL of the cDNA synthesis reaction mixture with 2.5 μL forward primer (final concentration 1.0 mM), 2.5 μL reverse primer (final concentration 1.0 mM), 10 μL of SYBR Premix Extaq Perfect Real Time (TAKARA Bio Inc., Japan) and water to a final volume of 20 μL. The qRT-PCR was performed using the program as essentially described in Nan et al. (2014) .
Data of phenotype were analyzed with the SPSS (Version 21.0) using one-way analysis of variance. Data of the expression of genes were analyzed with the SPSS (Version 21.0) using the Student’s t-test analysis.
We thank Professor Yaoguang Liu from South China Agricultural University for providing the vector pYLCRISPR/Cas9P35S-B. We thank Dr. Kan Wang, Iowa State University of Science and Technology for providing the vector pTF101.1 and Agrobacterium strain EHA101.
The research was supported by the National Genetically Modified Organisms Breeding Major Projects (2016ZX08004–005). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets and materials generated and analyzed during the current study are available from the corresponding author on reasonable request.
DC and L-SPT conceived and designed the experiments; AB and HC constructed the vectors; AB, LC and XZ1 performed the Agrobacterium-mediated transformation; AB, SC and QH performed the mutation analysis; WG performed the qRT-PCR analysis; DQ, ZS, ZY and SY performed the investigation of phenotypic parameter; CZ analyzed the data; XL, FK and BL generated the GmmiR156b-OX and GmSPL9b-OX plants; XZ2, L-SPT, DC wrote and revised the paper. All the authors read and finalized the version before submission.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 21.Sun Z, Su C, Yun J, Jiang Q, Wang L, Wang Y, Cao D, Zhao F, Zhao Q, Zhang M, et al. Genetic improvement of the shoot architecture and yield in soybean plants via the manipulation of GmmiR156b. Plant Biotechnol J. 2018; doi.org/10.1111/pbi.12946.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.