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.

Development and validation of InDel markers for identification of QTL underlying flowering time in soybean

Soybean [Glycine max(L.) Merrill] is a major plant source of protein and oil. An accurate and well-saturated molecular linkage map is a prerequisite for forward genetic studies of gene function and for modern breeding for many useful agronomic traits. Next-generation sequence data available in public databases provides valuable information and offers new insights for rapid and efficient development of molecular markers. In this study, we attempted to show the feasibility and facility of using genomic resequencing data as raw material for identifying putative In Del markers. First, we identified 17,613 In Del sites among 56 soybean accessions and obtained 12,619 primer pairs. Second, we constructed a genetic map with a random subset of 2841 primer pairs and aligned 300 polymorphic markers with the 20 consensus linkage groups(LG). The total genetic distance was 2347.3 c M and the number of mapped markers per LG ranged from 10 to 23 with an average of 15 markers. The largest and smallest genetic distances between adjacent markers were 52.3 c M and 0.1 cM, respectively. Finally, we validated the genetic map constructed by newly developed In Del markers by QTL analysis of days to flowering(DTF) under different environments. One major QTL(qDTF4) and four minor QTL(qDTF20, qDTF13, qDTF12,and q DTF11) on 5 LGs were detected. These results demonstrate the utility of the In Del markers developed in this work for map-based cloning and molecular breeding in soybean.

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.

FT5a interferes with the Dt1-AP1 feedback loop to control flowering time and shoot determinacy in soybean

Flowering time and stem growth habit determine inflorescence architecture in soybean, which in turn influences seed yield. Dt1, a homolog of Arabidopsis TERMINAL FLOWER 1(TFL1), is a major controller of stem growth habit, but its underlying molecular mechanisms remain unclear.Here, we demonstrate that Dt1 affects node number and plant height, as well as flowering time,in soybean under long-day conditions. The b ZIP transcription factor FDc1 physically interacts with Dt1, and the FDc1-Dt1 complex directly represses the expression of APETALA1(AP1). We propose that FT5 a inhibits Dt1 activity via a competitive interaction with FDc1 and directly upregulates AP1. Moreover, AP1 represses Dt1 expression by directly binding to the Dt1 promoter, suggesting that AP1 and Dt1 form a suppressive regulatory feedback loop to determine the fate of the shoot apical meristem. These findings provide novel insights into the roles of Dt1 and FT5 a in controlling the stem growth habit and flowering time in soybean, which determine the adaptability and grain yield of this important crop.

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.