Multiplex CRISPR/Cas9-mediated knockout of soybean LNK2 advances flowering time

• Published in: The Crop journal 2021-08, Vol.9 (4), p.767-776
• Main Authors: Li, Zhaobo, Cheng, Qun, Gan, Zhuoran, Hou, Zhihong, Zhang, Yuhang, Li, Yongli, Li, Haiyang, Nan, Haiyang, Yang, Cen, Chen, Linnan, Lu, Sijia, Shi, Wenqian, Chen, Liyu, Wang, Yanping, Fang, Chao, Kong, Liping, Su, Tong, Li, Shichen, Kou, Kun, Wang, Lingshuang, Kong, Fanjiang, Liu, Baohui, Dong, Lidong
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; Flowering time; Genome editing; LNK2; Soybean
• ISSN: 2214-5141; 2214-5141
• DOI: 10.1016/j.cj.2020.09.005

Flowering time is an important agronomic trait for soybean yield and adaptation. However, the genetic basis of soybean adaptation to diverse latitudes is still not clear. Four NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 2 (LNK2) homeologs of Arabidopsis thaliana LNK2 were identified in soybean. Three single-guide RNAs were designed for editing the four LNK2 genes. A transgene-free homozygous quadruple mutant of the LNK2 genes was developed using the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Under long-day (LD) conditions, the quadruple mutant flowered significantly earlier than the wild-type (WT). Quantitative real-time PCR (qRT-PCR) revealed that transcript levels of LNK2 were significantly lower in the quadruple mutant than in the WT under LD conditions. LNK2 promoted the expression of the legume-specific E1 gene and repressed the expression of FT2a. Genetic markers were developed to identify LNK2 mutants for soybean breeding. These results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four LNK2 genes shortens flowering time in soybean. Our findings identify novel components in flowering-time control in soybean and may be beneficial for further soybean breeding in high-latitude environments.