CRISPR/Cas9‐mediated gene editing of GmJAGGED1 increased yield in the low‐latitude soybean variety Huachun 6

The number of seeds per pod (NSPP) is one of the critical components that affect soybean yield (Liu et al., 2020). Ln, which encodes GmJAGGED1 (GmJAG1), is a major locus regulating NSPP and leaflet shape in soybean (Fang et al., 2013; Jeong et al., 2012). A single base pair substitution of the Ln locus from guanine (G) to cytosine (C) led to an amino acid change in the conserved EAR motif of GmJAG1, resulting in the loss of function of GmJAG1 and ln (Fang et al., 2013; Jeong et al., 2012). ln can significantly increase NSPP (Fang et al., 2013) and has been widely used in soybean breeding. By introducing ln into Kedou 1, the yield was increased by approximately 8% to 10% and resulted in a new high‐yield variety Kedou 17 (Liu et al., 2020).

In order to breed the superior varieties for soybean in the tropical and subtropical regions, we intend to fulfil the advantage of the beneficial ln locus. For that purpose, we analysed the distribution of the natural ln allele in soybean populations. Among the total of more than 3800 accessions that have been re‐sequenced (Fang et al., 2017; Torkamaneh et al., 2021; Zhou et al., 2015), ln widely exists in the modern elite lines and less frequently exists in landraces (Fig. 1a). Interestingly, ln was hardly found in wild soybean (Glycine soja). The nucleotide diversity around the ln locus was extremely low, in strong contrast to Ln (Fig. 1b,c). This result is consistent with the previous report that ln is located in a selection sweep (Fang et al., 2013) and indicates the ln allele has been favourably selected for the modern soybean breeding practices, which leads to an enrichment of ln in modern soybean lines.

Figure 1.

CRISPR/Cas9 knockout of GmJAG1 increased yield in the low‐latitude soybean Huachun 6. (a) Allele frequency distribution of Ln and ln within wild, landrace and elite lines (top), and populations from the high and low latitudes (bottom). (b) The ratio of π between populations carrying Ln and ln across the 1‐Mb genomic region of GmJAG1. The black vertical line indicates GmJAG1. (c) The SNP density of the 1‐Mb genomic region of GmJAG1 from populations carrying Ln and ln. (d) Top, the gene exon–intron structure of GmJAG1 and GmJAG2. The blue vertical lines indicate the position of gRNAs; middle, the alignment of guide RNAs and target genomic sequences; bottom, the mutations of GmJAG1 and GmJAG2 were verified by Sanger sequencing. (e) The trifoliate leaves and pods of Huachun 6 and gmjag. Bar = 10 cm. (f) and (g) The percentages of 1‐seeded to 4‐seeded pods from summer (f) and spring (g) trials. P indicates a t‐test probability (n = 15). (h) The agronomic traits of Huachun 6 and gmjag from the summer and spring trials. Values are shown in mean ± s.e.m. (n = 15). The t‐test P values are *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Among the 173 re‐sequenced accessions collected from south and Southeast Asia and Brazil (Fang et al., 2017; Torkamaneh et al., 2021; Zhou et al., 2015), we did not find ln. In order to find ln from the low‐latitude soybean varieties, we collected an additional 210 accessions from south China and sequenced GmJAG1 by Sanger sequencing. However, ln was still not found, indicating that ln does not exist in the soybean germplasms that originated from low latitudes (Fig. 1a). Soybean is hypersensitive to photoperiod, which limits the intercross breeding between the varieties from different latitudes. Thus, it would be highly challenging to create ln‐carrying varieties in low latitudes by intercrossing between ln from high latitudes and varieties from low latitudes.

Given the nature that ln is the mutation of GmJAG1, it would be feasible to introduce the ln‐related beneficial traits into the varieties of low latitudes by mutating GmJAG1. For that, we employed the CRISPR/Cas9‐based genome‐editing technology and edited GmJAG1 in a low‐latitude variety Huachun 6, which is a spring soybean cultivar and has been certified by the Chinese government (Fig. 1d). Two guide RNAs were designed to target GmJAG1 (Fig. 1d). We obtained two independent T₀ lines carrying heterozygous mutations of GmJAG1. Due to the high sequence similarity between GmJAG1 and GmJAG2, GmJAG2 was also edited (Fig. 1d). From the T1 segregation population, we identified a CAS9‐free homozygous mutant, which was named as gmjag. Sanger sequencing showed that the start codons of GmJAG1 and GmJAG2 were lost in gmjag, in addition to the small deletions on the second exons of both genes (Fig. 1d). This plant and its offspring were used for further trait analysis. Consistent with the varieties carrying ln, gmjag showed narrow leaflets (Fig. 1e).

To evaluate the performance of gmjag, we conducted two field trials at Guangzhou experimental stations (N23°15′, E113°34′) in the summer of 2020 and the spring of 2021, respectively. The growth period, plant height, number of branches, number of nodes and number of total pods were not changed in gmjag compared with Huachun 6. Interestingly, we observed 4‐seeded pods in gmjag plants, whereas Huachun 6 did not develop any 4‐seeded pods (Fig. 1e). Additionally, the percentage of 3‐seeded pods was significantly increased in gmjag, whereas that of 2‐seeded pods was significantly decreased (Fig. 1f,g). The average NSPP and total seeds per plant of gmjag were significantly increased compared with Huachun 6. Although the single seed weight of gmjag was lower than that of Huachan 6 in the summer trial, no significant difference was observed in the spring trial (Fig. 1h). The yield of gmjag was increased by 8.81% and 8.67% in the spring and the summer trial, respectively. These results indicate that CRISPR/Cas9 knockout of GmJAG1 is effective in introducing ln‐related beneficial traits into the varieties in low latitudes.

CRISPR/Cas9 technology has been widely proposed as a powerful tool for rapid crop improvement. This work not only created new superior soybean germplasm by CRISPR/Cas9‐mediated gene editing but also provided a template for utilization of CRISPR/Cas9 technology in legume crop breeding.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

HN and LG designed the research; ZC, PX, YC, QM and TL performed the experiments; ZC and PX performed data analyses and ZC and LG wrote the manuscript.

Cai, Z. , Xian, P. , Cheng, Y. , Ma, Q. , Lian, T. , Nian, H. and Ge, L. (2021) CRISPR/Cas9‐mediated gene editing of GmJAGGED1 increased yield in the low‐latitude soybean variety Huachun 6. Plant Biotechnol. J., 10.1111/pbi.13673