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

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(q RT-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 FT2 a. 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.

Agronomical selection on loss-of-function of GIGANTEA simultaneously facilitates soybean salt tolerance and early maturity

Salt stress and flowering time are major factors limiting geographic adaptation and yield productivity in soybean(Glycine max).Although improving crop salt tolerance and latitude adaptation are essential for efficient agricultural production,whether and how these two traits are integrated remains largely unknown.Here,we used a genome-wide association study to identify a major salt-tolerance locus controlled by E2,an ortholog of Arabidopsis thaliana GIGANTEA(GI).Loss of E2 function not only shortened flowering time and maturity,but also enhanced salt-tolerance in soybean.E2 delayed soybean flowering by enhancing the transcription of the core flowering suppressor gene E1,thereby repressing Flowering Locus T(FT)expression.An E2 knockout mutant e2^(CR) displayed reduced accumulation of reactive oxygen species(ROS)during the response to salt stress by releasing peroxidase,which functions in ROS scavenging to avoid cytotoxicity.Evolutionary and population genetic analyses also suggested that loss-of-function e2 alleles have been artificially selected during breeding for soybean adaptation to high-latitude regions with greater salt stress.Our findings provide insights into the coupled selection for adaptation to both latitude and salt stress in soybean;and offer an ideal target for molecular breeding of early-maturing and salt-tolerant cultivars.