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.

GIGANTEA orthologs,E2 members,redundantly determine photoperiodic flowering and yield in soybean

Soybean(Glycine max L.)is a typical photoperiodsensitive crop,such that photoperiod determines its flowering time,maturity,grain yield,and phenological adaptability.During evolution,the soybean genome has undergone two duplication events,resulting in about 75%of all genes being represented by multiple copies,which is associated with rampant gene redundancy.Among duplicated genes,the important soybean maturity gene E2 has two homologs,E2-Like a(E2La)and E2-Like b(E2Lb),which encode orthologs of Arabidopsis GIGANTEA(GI).Although E2 was cloned a decade ago,we still know very little about its contribution to flowering time and even less about the function of its homologs.Here,we generated single and double mutants in E2,E2La,and E2Lb by genome editing and determined that E2 plays major roles in the regulation of flowering time and yield,with the two E2 homologs depending on E2 function.At high latitude regions,e2 single mutants showed earlier flowering and high grain yield.Remarkably,in terms of genetic relationship,genes from the legume-specific transcription factor family E1 were epistatic to E2.We established that E2 and E2-like proteins form homodimers or heterodimers to regulate the transcription of E1 family genes,with the homodimer exerting a greater function than the heterodimers.In addition,we established that the H3 haplotype of E2 is the ancestral allele and is mainly restricted to low latitude regions,from which the loss-of-function alleles of the H1 and H2haplotypes were derived.Furthermore,we demonstrated that the function of the H3 allele is stronger than that of the H1 haplotype in the regulation of flowering time,which has not been shown before.Our findings provide excellent allelic combinations for classical breeding and targeted gene disruption or editing.

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.

Soybean AP1 homologs control flowering time and plant height

Flowering time and plant height are key agronomic traits that directly affect soybean(Glycine max)yield.APETALA1(AP1)functions as a class A gene in the ABCE model for floral organ development,helping to specify carpel,stamen,petal,and sepal identities.There are four AP1 homologs in soybean,all of which are mainly expressed in the shoot apex.Here,we used clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein 9 technology to generate a homozygous quadruple mutant,gmap1,with loss-of-function mutations in all four GmAP1 genes.Under short-day(SD)conditions,the gmap1 quadruple mutant exhibited delayed flowering,changes in flower morphology,and increased node number and internode length,resulting in plants that were taller than the wild type.Conversely,overexpression of GmAP1a resulted in early flowering and reduced plant height compared to the wild type under SD conditions.The gmap1 mutant and the overexpression lines also exhibited altered expression of several genes related to flowering and gibberellic acid metabolism,thereby providing insight into the role of GmAP1 in the regulatory networks controlling flowering time and plant height in soybean.Increased node number is the trait with the most promise for enhancing soybean pod number and grain yield.Therefore,the mutant alleles of the four AP1 homologs described here will be invaluable for molecular breeding of improved soybean yield.

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.

用户侧用电需求响应行为研究

随着各种分布式电源和新型用电设备的日益推广以及电力用户对需求个性化要求的提升,智能用电的理念及其技术发展体系应运而生。为获得经济、环境效益的同时提升电能服务质量,研究用电行为并主动整合用户侧资源、优化用能调度已成为一项积极而有效的措施。提出了一种基于用户行为学的家庭负荷,对电力用户侧的需求响应行为展开研究。为了揭示用户用电量对电价、气象、日类型等相关变化因素的响应程度,提出了一种需求响应综合模型。该模型实现了用户在分时电价下的需求响应行为规律的模拟,可为电力公司制定需求侧管理策略提供基础性指导。

A recent retrotransposon insertion of J caused E6 locus facilitating soybean adaptation into low latitude

Soybean(Glycine max) is an important legume crop that was domesticated in temperate regions.Soybean varieties from these regions generally mature early and exhibit extremely low yield when grown under inductive short-day(SD) conditions at low latitudes. The long-juvenile(LJ) trait, which is characterized by delayed flowering and maturity,and improved yield under SD conditions, allowed the cultivation of soybean to expand to lower latitudes. Two major loci control the LJ trait: J and E6. In the current study, positional cloning, sequence analysis, and transgenic complementation confirmed that E6 is a novel allele of J, the ortholog of Arabidopsis thaliana EARLY FLOWERING 3(ELF3). The mutant allele e6^(PG), which carries a Ty1/Copia-like retrotransposon insertion, does not suppress the legume-specific flowering repressor E1, allowing E1 to inhibit Flowering Locus T(FT) expression and thus delaying flowering and increasing yields under SD conditions. The e6^(PG)allele is a rare allele that has not been incorporated into modern breeding programs.The dysfunction of J might have greatly facilitated the adaptation of soybean to low latitudes. Our findings increase our understanding of the molecular mechanisms underlying the LJ trait and provide valuable resources for soybean breeding.