Target site selection, construction, and confirmation of the target sites in soybean hairy roots
In order to identify the ortholog of AtLHY and AtCCA1 in soybean, we performed protein sequence alignment and identified four CCA1/LHY orthologs in soybean. Phylogenetic analysis showed that the four CCA1/LHY orthologs are closer to AtLHY than AtCCA1. Thus, the four CCA1/LHY orthologs was named GmLHY1a (Glyma.16G017400), GmLHY1b (Glyma.07G048500), GmLHY2a (Glyma.19G260900), and GmLHY2b (Glyma.03G261800) (Figure S1). To study the function of the four GmLHY genes in soybean, four target adaptors were used, including target 1/2 for targeting the GmLHY2a and GmLHY2b genes, and target 3/4 for targeting the GmLHY1a and GmLHY1b genes (Fig.1A). Target 1 is present in the second and third exon of the GmLHY2b and GmLHY2a genes, respectively; target 2 is present in the fifth and sixth exon of the GmLHY2b and GmLHY2a genes, respectively; target 3 is present in the first exon of GmLHY1a and GmLHY1b; and target 4 is present in the fifth exon of GmLHY1a and GmLHY1b in soybean (Fig. 1A). The CRISPR vector encodes Cas9 and was driven by the CaMV35S promoter and four gRNAs driven by the Arabidopsis U3b, U3d, U6-1, and U6-29 promoters, respectively (Fig. 1B, C).
In order to test whether the CRISPR/Cas9 construct could properly edit these genes in transgenic soybean plants, we first tested the construct in transgenic soybean hairy roots using A. rhizogenes K599 (Figure S2A). The transgenic soybean hairy roots were generated by high-efficiency Agrobacterium rhizogenes-mediated transformation . When the hairy roots generated at the infection site were approximately 2 cm long, they were used for genotype detection. The genotype of the transgenic hairy roots was detected by PCR using Cas9 gene-specific primers and GmLHY gene-specific primers. We detected mobility-shifted bands in six DNA-bulked samples when the Cas9 gene-specific primers were utilized. The result showed that there were five transgenic lines with the Cas9 gene product (Cas9 gene-positive) (Figure S2B). Sequencing analysis of the GmLHY genes showed that the Cas9 gene-positive lines (R1–R5) produced superimposed peaks in the target 1/3 site, while the target 2/4 site was unchanged (Figure S2C, Table S1). Together, these results indicated that the transgene-encoded Cas9 and gRNAs were able to efficiently induce double-strand breaks at the target 1/3 sites in the GmLHY genes.
Transgene-free homozygous quadruple mutant of GmLHY in soybean
We next performed stable soybean transformation and obtained 19 independent T0 transgenic lines with the section for the Cas9 gene product (Cas9 gene-positive) (Figure S3A). Sequencing analysis showed that the T0-7 line was a heterozygous quadruple mutant of GmLHY that might possess a 2-bp deletion in GmLHY2b/2a/1b-target1/3 and a 1-bp deletion in GmLHY2a-target3 (Figure S3B-E; Table S2). In order to use the mutants in crop breeding, we sought homozygous quadruple mutants of the GmLHY line without the transgene and screened the T1 plants derived from the T0 transgenic lines. Fortunately, we obtained eight T1 plants derived from T0-7 that lacked the Cas9 gene (Fig. 2A, B), and only one line (T1-15) was a transgene-free homozygous quadruple mutant of GmLHY (Fig. 2C–F; Table S2). Sequencing analysis showed that the quadruple mutant of GmLHY had a 2-bp deletion in GmLHY2b/2a/1b-target1/3 and a 1-bp deletion in GmLHY1a-target3 (Fig. 2C–2F), resulting in frame-shift mutations in the GmLHY genes (Fig. 2G).
The expression level of GmLHY in the quadruple mutant and WT
LHY/CCA1 are key components of the circadian clock and participate in the temporal organization of biological activities and the regulation of gene expression [16, 17, 21]. Previous studies have shown that the expression level of LHY/CCA1 was much higher in the morning than in the night . However, the expression pattern of GmLHY genes in the quadruple mutant of GmLHY is not known. The diurnal circadian rhythm of GmLHY gene expression in the quadruple mutant of GmLHY was analyzed by quantitative real-time PCR (qRT-PCR) under inductive long-day (LD) conditions. The result showed that GmLHY1a, GmLHY1b, GmLHY2a, and GmLHY2b were highly up-regulated in WT, and the highest expression was detected at 0 h and 24 h after dawn (Fig. 3A–D). However, the expression of GmLHY genes was lower in the quadruple mutant of GmLHY than WT (Fig. 3A–D). These results showed that the expression of the four GmLHY genes was significantly decreased in the quadruple mutant of GmLHY.
The quadruple mutant of GmLHY reduces soybean plant height and shortens internodes
To examine the loss function of GmLHY, the phenotypes of the T2-generation transgene-free quadruple mutant and WT plants were observed. We found that the plant height of the quadruple mutant was significantly lower than WT under LD conditions for 20 days after emergence (DAE) (Fig. 4A, B). Subsequently, we examined the node number and internodal length, as these impact plant height [13, 15]. As indicated in Fig. 4C and 4D, the node number did not change, while the internodal length was significantly shorter in the quadruple mutant than WT. These results suggested that the dwarfed plant height of the quadruple mutant was caused by a shorter length. We also analyzed the plant height of the quadruple mutant and WT from 20 to 35 DAE (Fig. 4E). The result showed that the height of the quadruple mutant of GmLHY was shorter from 20 to 35 DAE.
The quadruple mutant of GmLHY is deficient in the GA biosynthesis pathway
Previous studies showed that GAs is one of the most important phytohormones determining plant height [41, 42]. To test whether GmLHY affects the GA biosynthesis pathway, the Gmlhy1a1b2a2b mutant and WT were treated with GA3 and Uni (uniconazole, a GA biosynthesis inhibitor). The results showed that exogenous GA3 could restore the Gmlhy1a1b2a2b mutant to the WT, and Uni treatment could reduce the plant height of the WT and Gmlhy1a1b2a2b mutant seedlings (Fig 5A, 5B). Endogenous GA3 levels from both the WT and Gmlhy1a1b2a2b mutant were determined using liquid chromatography–mass spectrometry (LC-MS). The results suggested that the levels of endogenous GA3 in Gmlhy1a1b2a2b were lower than in WT (Figure 5C). These findings indicated that the Gmlhy1a1b2a2b mutant has a low active gibberellin level and that it is a GA biosynthesis-deficient mutant.
Expression analysis of GA metabolic pathway-related genes in the quadruple mutant of GmLHY and WT plants
Next, qRT-PCR was performed to measure the relative expression of genes that are known to participate in GA biosynthesis, such as GA-20 oxidase (GmGA1, Glyma.09G149200; GmGA2, Glyma.20G153400), copalyl pyrophosphate synthase (GmCPS2, Glyma.19G157000), ent-kaurene synthase (GmDW1, Glyma.08G163900), and GA-responsive genes (GmGR2, Glyma.20G230600; GmGR8, Glyma.11G216500)  in WT and the quadruple mutant of GmLHY. Compared with the WT plants, these genes showed significantly decreased expression in the quadruple mutant of GmLHY (Fig. 6A–F). Our findings suggested that GmLHY might positively regulate the expression of these GA biosynthesis and GA responsive genes, thereby limiting soybean plant height.
Development of genetic markers and inheritance of quadruple mutant alleles
Genetic markers provide a critical and effective means of identifying mutant alleles for molecular-assisted studies and could possibly accelerate the genotyping procedure in future generations . Therefore, we developed three dCAPs (Derived Cleaved Amplified Polymorphic Sequences) markers to identify the Gmlhy1a1b2a2b mutant alleles (Fig. 7A). For the genotyping of the Gmlhy1a1b2a2b mutants, PCR amplifications were performed using GmLHY-specific and dCAPs-specific primer pairs. The amplified products of GmLHY2b, GmLHY2a, and GmLHY1b on the mutant genomic DNA templates, but not on the WT genomic DNA templates, could be cleaved by the restriction endonuclease MspI (Fig. 7B). Additionally, the amplified products of GmLHY1a on the mutant genomic DNA templates, but not on the WT genomic DNA templates, could be cleaved by restriction endonuclease RspRSII (Fig. 7B). These results confirmed that the three dCAPs markers of GmLHY could be used for the genotyping of Gmlhy1a1b2a2b mutants and have further prospect in molecular breeding studies.