Target site selection, construction and confirmation of 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, GmLCL1 (Glyma.03G261800),, GmLCL2 (Glyma.19G260900),, GmLCL3 (Glyma.16G017400),, and GmLCL4 (Glyma.07G048500) in soybean. Phylogenetic analysis showed that GmLCL proteins closer to AtLHY than AtCCA1 (Figure S1). To study their function of the four GmLCL genes in soybean, four target adaptors, target 1/2 were selected for targeting GmLCL1 and GmLCL2 genes, target 3/4 were selected for targeting GmLCL3 and GmLCL4 genes (Fig.1A). The target 1 in the second and third exon of GmLCL1 and GmLCL2 genes respectively, target 2 in the fifth and sixth exon of GmLCL1 and GmLCL2 genes respectively, and the target 3 in the first exon of GmLCL3 and GmLCL4, the target 4 in the fifth exon of GmLCL3 and GmLCL4 in soybean (Fig. 1A). The CRISPR vector used encodes Cas9 driven by the CaMV35S promoter and four gRNAs driven by the Arabidopsis U3b, U3d, U6–1 and U6–29 promoter, respectively (Fig. 1B, C).
In order to test whether the CRISPR/Cas9 construct could edit properly these genes in transgenic soybean plants, we first tested the construct in transgenic soybean hairy roots usingA. 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, the hairy roots were used for genotype detection. The genotype of transgenic hairy roots was detected by PCR using Cas9 gene-specific primers and GmLCL genes-specific primers. We detected mobility- shifted bands in six DNA bulked samples when specific primers of Cas9 gene-specific were utilized. The result showed that five transgenic lines with the Cas9 gene product (Cas9 gene-positive) (Figure S2B). Sequencing analysis of GmLCL genes showed that the Cas9 gene-positive lines (R1-R5) produced superimposed peaks in target1/3 site, while the target 2/4 site no changed (Figure S2C, Table S1). Together, these results indicated that the transgene-encoded Cas9 and gRNAs were able to efficiently induce double-strand breaks at target 1/3 sites in GmLCL genes.
Transgene-free homozygous quadruple mutant of GmLCL in soybean
We next performed stable soybean transformation and obtained nineteen 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 GmLCL, and might have a 2-bp deletion in the GmLCL1/2/4-target1/3 and might have a 1-bp deletion in the GmLCL3-target3 (Figure S3B-E; Table S2). In order to apply the mutants to crop breeding, we sought homozygous quadruple mutant of GmLCL line without transgene and screened T1 plants derived from the T0 transgenic lines. Luckily, we obtained eight T1 plants derived from T0–7 showed the absence of Cas9 gene (Fig. 2A, B), and only one line (T1–15) was transgene-freehomozygous quadruple mutant of GmLCL (Fig. 2C-F; Table S2). Sequencing analysis showed that quadruple mutant of GmLCL had a 2-bp deletion in the GmLCL1/2/4-target1/3, and had a 1-bp deletion in the GmLCL3-target3 (Fig. 2C–2F), resulting in frame-shift mutations in GmLCL1/2/3/4 genes (Fig. 2G).
The expression level of the GmLCLs in quadruple mutant and WT
LHY/CCA1 are key components of the circadian clock, which participate the temporal organization of biological activities and the regulation of gene expression [16, 17, 21]. Previous studies had shown the expression level of LHY/CCA1 were much higher in the morning than in the night . However, the expression pattern of GmLCL genes in quadruple mutant of GmLCL is still unclear. The diurnal circadian rhythm of GmLCL genes expression in quadruple mutant of GmLCL was analyzed by quantitative real-time PCR (qRT-PCR) under inductive long-day (LD) conditions. The result showed that GmLCL1, GmLCL2, GmLCL3 and GmLCL4 were highly up-regulated in WT and the highest expression at 0 h and 24 h after dawn (Fig. 3A-D). But, the expression of GmLCL genes were lower in quadruple mutant of GmLCL than WT (Fig. 3A-D). These results showed that the expression of four GmLCL genes was significant decreased in quadruple mutant of GmLCL.
The quadruple mutant of GmLCL reduce soybean plant height and shorten internode
To examine the loss function of GmLCLs, the phenotype of T2-generation transgene-free quadruple mutant and WT plants were observed. We found that the plant height of quadruple mutant was significantly lower than WT under LD conditions for 20 days after emergence (DAE) (Fig. 4A, B). Subsequently, we examined that the node number andinternodal length, which could affect plant height [13, 15]. As showed in Fig. 4C and 4D, the node number had no change, while the internodal length was significantly shorter in quadruple mutant than WT. These results showed that the dwarf of quadruple mutant plant height was caused by shorter internodal length. We also analysed the plant height of quadruple mutant and WT from 20 to 35 DAE (Fig. 4E). The result showed that the quadruple mutant of GmLCL shortens plant height from 20 to 35 DAE.
Expression analysis of GA metabolic pathway‑related genes in quadruple mutant of GmLCL and WT plants
Previous studies showed that gibberellins (GAs) are one of the most important phytohormones determining plant height in the plants [41, 42]. To address how GmLCLs affects plant height, we performed qRT-PCR in WT and quadruple mutant of GmLCL to measure the relative expression of genes that are known to take part 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) . Compared with the WT plants these genes showed significantly decreased expression in quadruple mutant of GmLCL (Fig. 5A–F). These results suggested that GmLCLs 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 are critical and effective method to identify mutant alleles for molecular-assisted studies, and the genetic markers could accelerate genotyping procedure in future generations . Therefore, we developed three dCAPs (Derived Cleaved Amplified Polymorphic Sequences) markers to identify the GmLCLs mutant alleles (Fig. 6A). To identify the genotyping of GmLCL mutants, PCR amplifications were performed using GmLCLs-specific and dCAPs-specific primer pairs, respectively. The amplified products of GmLCL1, GmLCL2 and GmLCL4 on mutant genomic DNA templates could be cleaved by restriction endonuclease MspI, but not on WT genomic DNA templates (Fig. 6B). Meanwhile, the amplified products of GmLCL3 on mutant genomic DNA templates could be cleaved by restriction endonuclease RspRSII, but not on WT genomic DNA templates (Fig. 6B). These results showed that three dCAPs markers of GmLCLs could be used to identify the genotyping of GmLCL mutants and used for molecular breeding studies.