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.

Parallel selection of distinct Tof5 alleles drove the adaptation of cultivated and wild soybean to high latitudes

Photoperiod responsiveness is a key factor limiting the geographic distribution of cultivated soybean and its wild ancestor.In particular,the genetic basis of the adaptation in wild soybean remains poorly understood.In this study,by combining whole-genome resequencing and genome-wide association studies we identified a novel locus,Time of Flowering 5(Tof5),which promotes flowering and enhances adaptation to high latitudes in both wild and cultivated soybean.By genomic,genetic and transgenic analyses we showed that Tof5 en-codes a homolog of Arabidopsis thaliana FRUITFULL(FUL).Importantly,further analyses suggested that different alleles of Tof5 have undergone parallel selection.The Tof5H1 allele was strongly selected by humans after the early domestication of cultivated soybean,while Tof5H2 allele was naturally selected in wild soybean,and in each case facilitating adaptation to high latitudes.Moreover,we found that the key flowering repressor E1 suppresses the transcription of Tof5 by binding to its promoter.In turn,Tof5 physically associates with the promoters of two important FLOWERING LOCUS T(FT),FT2a and FT5a,to upregulate their transcription and promote flowering under long photoperiods.Collectively,ourfindings provide insights into how wild soybean adapted to high latitudes through natural selection and indicate that cultivated soybean underwent changes in the same gene but evolved a distinct allele that was artificially selected after domestication.

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.

Elimination of an unfavorable allele conferring pod shattering in an elite soybean cultivar by CRISPR/Cas9

Pod shattering can lead to devastating yield loss of soybean and has been a negatively selected trait in soybean domestication and breeding.Nevertheless,a significant portion of soybean cultivars are still pod shattering-susceptible,limiting their regional and climatic adaptabilities.Here we performed genetic diagnosis on the shattering-susceptible trait of a national registered cultivar,Huachun6(HC6),and found that HC6 carries the susceptible genotype of a candidate Pod dehiscence 1(PDH1)gene,which exists in a significant portion of soybean cultivars.We next performed genome editing on PDH1 gene by clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9(Cas9).In T2 progenies,several transgene-free lines with pdh1 mutations were characterized without affecting major agronomic traits.The pdh1 mutation significantly improved the pod shattering resistance which is associated with aberrant lignin distribution in inner sclerenchyma.Our work demonstrated that precision breeding by genome editing on PDH1 holds great potential for precisely improving pod shattering resistance and adaptability of soybean cultivars.