GmAP1d regulates flowering time under long-day photoperiods in soybean

Flowering time is important for adaptation of soybean(Glycine max)to different environments.Here,we conducted a genome-wide association study of flowering time using a panel of 1490 cultivated soybean accessions.We identified three strong signals at the qFT02-2 locus(Chr02:12037319–12238569),which were associated with flowering time in three environments:Gongzhuling,Mengcheng,and Nanchang.By analyzing linkage disequilibrium,gene expression patterns,gene annotation,and the diversity of variants,we identified an AP1 homolog as the candidate gene for the qFT02-2 locus,which we named GmAP1d.Only one nonsynonymous polymorphism existed among 1490 soybean accessions at position Chr02:12087053.Accessions carrying the Chr02:12087053-T allele flowered significantly earlier than those carrying the Chr02:12087053-A allele.Thus,we developed a cleaved amplified polymorphic sequence(CAPS)marker for the SNP at Chr02:12087053,which is suitable for marker-assisted breeding of flowering time.Knockout of GmAP1d in the‘Williams 82’background by gene editing promoted flowering under long-day conditions,confirming that GmAP1d is the causal gene for qFT02-2.An analysis of the region surrounding GmAP1d revealed that GmAP1d was artificially selected during the genetic improvement of soybean.Through stepwise selection,the proportion of modern cultivars carrying the Chr02:12087053-T allele has increased,and this allele has become nearly fixed(95%)in northern China.These findings provide a theoretical basis for better understanding the molecular regulatory mechanism of flowering time in soybean and a target gene that can be used for breeding modern soybean cultivars adapted to different latitudes.

J-family genes redundantly regulate flowering time and increase yield in soybean

Soybean(Glycine max)is a short-day crop whose flowering time is regulated by photoperiod.The longjuvenile trait extends its vegetative phase and increases yield under short-day conditions.Natural variation in J,the major locus controlling this trait,modulates flowering time.We report that the three J-family genes influence soybean flowering time,with the triple mutant Guangzhou Mammoth-2 flowering late under short days by inhibiting transcription of E1-family genes.J-family genes offer promising allelic combinations for breeding.

Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

Two soybean homologues of TERMINAL FLOWER 1 control flowering time under long day conditions

Flowering time is a key agronomic trait that directly affect the adaptation and yield of soybean.After whole genome duplications,about 75%of genes being represented by multiple copies in soybean.There are four TERMINAL FLOWER 1(TFL1)genes in soybean,and the TFL1b(Dt1)has been characterized as the determinant of stem growth habit.The function of other TFL1 homologs in soybean is still unclear.Here,we generated knockout mutants by CRISPR/Cas9 genome editing technology and found that the tfl1c/tfl1d double mutants flowered significantly earlier than wild-type plants.We investigated that TFL1c and TFL1d could physically interact with the b ZIP transcription factor FDc1 and bind to the promoter of APETALA1a(AP1a).RNA-seq and q RT-PCR analyses indicated that TFL1c and TFL1d repressed the expressions of the four AP1 homologs and delayed the flowering time in soybean.The two genes play important roles in the regulation of flowering time in soybean and mainly act as the flowering inhibitors under long-day conditions.Our results identify novel components in the flowering-time regulation network of soybean and will be invaluable for molecular breeding of improved soybean yield.

A differentially methylated region of the ZmCCT10 promoter affects flowering time in hybrid maize

Flowering time(FT) is a key maize domestication trait, variation in which allows maize to grow in a wide range of latitudes. Although previous studies have investigated the genetic control of FT-related traits per se, few studies of FT hybrid performance have been published. We characterized the genomic architecture associated with hybrid performance for FT in a hybrid panel by testcrossing Chang 7–2 with 328Ye478 × Qi319 recombinant inbred lines(RILs). We identified 11 quantitative trait loci(QTL) for hybrid performance in FT-related traits, including a major QTL qFH10 that controls hybrid performance and heterosis in a summer maize-growing region. However, this locus acts in regulating FT traits per se only in a spring maize-growing region. We validated ZmCCT10 as a candidate gene for qFH10 and found that differences between hybrids and their parental lines in DNA methylation in the differentially methylated region(DMR, –700 to –1520) of the ZmCCT10 promoter affected gene expression pattern and thereby FT in the summer maize-growing region.

Accession-specific flowering time variation in response to nitrate fluctuation in Arabidopsis thaliana

Flowering time,a key transition point from vegetative to reproductive growth,is regulated by an intrinsic complex of endogenous and exogenous signals including nutrient status.For hundreds of years,nitrogen has been well known to modulate flowering time,but the molecular genetic basis on how plants adapt to ever-changing nitrogen availability remains not fully explored.Here we explore how Arabidopsis natural variation in flowering time responds to nitrate fluctuation.Upon nitrate availability change,we detect accession-and photoperiod-specific flowering responses,which also feature a accession-specific dependency on growth traits.The flowering time variation correlates well with the expression of floral integrators,SOC1 and FT,in an accession-specific manner.We find that gene expression variation of key hub genes in the photoperiod-circadian-clock(GI),aging(SPLs)and autonomous(FLC)pathways associates with the expression change of these integrators,hence flowering time variation.Our results thus shed light on the molecular genetic mechanisms on regulation of accession-and photoperiod-specific flowering time variation in response to nitrate availability.

Mapping QTL for flowering time-related traits under three plant densities in maize

Flowering time is an indicator of adaptation in maize and a key trait for selection in breeding.The genetic basis of flowering time in maize,especially in response to plant density,remains unclear.The objective of this study was to identify maize quantitative trait loci(QTL)associated with flowering time-related traits that are stably expressed under several plant densities and show additive effects that vary with plant density.Three hundred recombinant inbred lines(RIL)derived from a cross between Ye 478 and Qi 319,together with their parents,were planted at three plant densities(90,000,120,000,and 150,000 plants ha^(-1))in four environments.The five traits investigated were days to tasseling(DTT),days to silking(DTS),days to pollen shed(DTP),interval between anthesis and silking(ASI),and interval between tasseling and anthesis(TAI).A high-resolution bin map was used for QTL mapping.In the RIL population,the DTT,DTS,and DTP values increased with plant density,whereas the ASI and TAI values showed negligible response to plant density.A total of 72 QTL were identified for flowering time-related traits,including 15 stably expressed across environments.Maize flowering time under different densities seems to be regulated by complex pathways rather than by several major genes or an independent pathway.The effects of some stable QTL,especially qDTT8-1 and qDTT10-4,varied with plant density.Fine mapping and cloning of these QTL will shed light on the mechanism of flowering time and assist in breeding earlymaturing maize inbred lines and hybrids.

CRISPR/Cas9-engineered mutation to identify the roles of phytochromes in regulating photomorphogenesis and flowering time in soybean

Soybean(Glycine max)responds to ambient light variation by undergoing multiform morphological alterations,influencing its yield potential and stability in the field.Phytochromes(PHYs)are plant-specific red(R)and far-red(FR)light photoreceptors mediating photomorphogenesis and photoperiodic flowering.As an ancient tetraploid,soybean harbors four PHYA,two PHYB,and two PHYE paralogs.Except for GmPHYA2/E4 and GmPHYA3/E3,which have been identified as photoperiod-dependent flowering repressors,the functions of GmPHYs are still largely unclear.We generated a series of individual or combined mutations targeting the GmPHYA or GmPHYB genes using CRISPR/Cas9 technology.Phenotypic analysis revealed that GmPHYB1 mediates predominantly R-light induced photomorphogenesis,whereas GmPHYA2/E4 and GmPHYA3/E3,followed by GmPHYA1 and GmPHYB2,function redundantly and additively in mediating FR light responses in seedling stage.GmPHYA2/E4 and GmPHYA3/E3,with weak influence from GmPHYA1 and GmPHYA4,delay flowering time under natural long-day conditions.This study has demonstrated the diversified functions of GmPHYAs and GmPHYBs in regulating light response,and provides a core set of phytochrome mutant alleles for characterization of their functional mechanisms in regulating agronomic traits of soybean.

Development of mutants with varying flowering times by targeted editing of multiple SVP gene copies in Brassica napus L.

Manipulation of flowering time to develop cultivars with desired maturity dates is fundamental in plant breeding.It is desirable to generate polyploid rapeseed(Brassica napus L.)germplasm with varying flowering time controlled by a few genes.In the present study,Bna SVP,a rapeseed homolog of the Arabidopsis SVP(Short Vegetative Phase)gene,was characterized and a set of mutants was developed using a CRISPR/Cas9-based gene-editing tool.A single construct targeting multiple sites was successfully applied to precisely mutate four copies of Bna SVP.The induced mutations in these copies were stably transmitted to subsequent generations.Homozygous mutants with loss-of-function alleles and free transgenic elements were generated across the four Bna SVP homologs.All mutant T_(1)lines tested in two environments(summer and winter growing seasons)showed early-flowering phenotypes.The decrease in flowering time was correlated with the number of mutated Bna SVP alleles.The quadruple mutants showed the shortest flowering time,with a mean decrease of 40.6%–50.7%in length relative to the wild type under the two growth conditions.Our study demonstrates the quantitative involvement of Bna SVP copies in the regulation of flowering time and provides valuable resources for rapeseed breeding.

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.

Expansion and expression diversity of FAR1/FRS-like genes provides insights into flowering time regulation in roses

Roses are important horticultural plants with enormous diversity in flowers and flowering behavior.However,molecular regulation of flowering time variation in roses remains poorly characterized.Here,we report an expansion of the FAR1/FRS-like genes that correlates well with the switch to prostrate-toerect growth of shoots upon flowering in Rosa wichuraiana‘Basye's Thornless'(BT).With the availability of the high-quality chromosome-level genome assembly for BT that we developed recently,we identified 91 RwFAR1/FRS-like genes,a significant expansion in contrast to 52 in Rosa chinensis‘Old Blush’(OB),a founder genotype in modern rose domestication.Rose FAR1/FRS-like proteins feature distinct variation in protein domain structures.The dispersed expansion of RwFAR1/FRS-like genes occurred specifically in clade I and II and is significantly associated with transposon insertion in BT.Most of the RwFAR1/FRS-like genes showed relatively higher expression level than their corresponding orthologs in OB.FAR1/FRS-like genes regulate light-signaling processes,shade avoidance,and flowering time in Arabidopsis thaliana.Therefore,the expansion and duplication of RwFAR1/FRS-like genes,followed by diversification in gene expression,might offer a novel leverage point for further understanding the molecular regulation of the variation in shoot-growth behavior and flowering time in roses.

QTL effects and epistatic interaction for flowering time and branch number in a soybean mapping population of Japanese×Chinese cultivars

Flowering time and branching type are important agronomic traits related to the adaptability and yield of soybean. Molecular bases for major flowering time or maturity loci, E1 to E4, have been identified. However, more flowering time genes in cultivars with different genetic backgrounds are needed to be mapped and cloned for a better understanding of flowering time regulation in soybean. In this study, we developed a population of Japanese cultivar（Toyomusume）×Chinese cultivar（Suinong 10） to map novel quantitative trait locus（QTL） for flowering time and branch number. A genetic linkage map of a F_2 population was constructed using 1 306 polymorphic single nucleotide polymorphism（SNP） markers using Illumina Soy SNP8 ki Select Bead Chip containing 7 189（SNPs）. Two major QTLs at E1 and E9, and two minor QTLs at a novel locus, qFT2_1 and at E3 region were mapped. Using other sets of F_2 populations and their derived progenies, the existence of a novel QTL of qFT2_1 was verified. qBR6_1, the major QTL for branch number was mapped to the proximate to the E1 gene, inferring that E1 gene or neighboring genetic factor is significantly contributing to the branch number.

GhAP1-D3 positively regulates flowering time and early maturity with no yield and fiber quality penalties in upland cotton

Flowering time(FTi)is a major factor determining how quickly cotton plants reach maturity.Early maturity greatly affects lint yield and fiber quality and is crucial for mechanical harvesting of cotton in northwestern China.Yet,few quantitative trait loci(QTLs)or genes regulating early maturity have been reported in cotton,and the underlying regulatory mechanisms are largely unknown.In this study,we characterized 152,68,and 101 loci that were significantly associated with the three key early maturity traits—FTi,flower and boll period(FBP)and whole growth period(WGP),respectively,via four genome-wide association study methods in upland cotton(Gossypium hirsutum).We focused on one major early maturity-related genomic region containing three single nucleotide polymorphisms on chromosome D03,and determined that GhAP1-D3,a gene homologous to Arabidopsis thaliana APETALA1(AP1),is the causal locus in this region.Transgenic plants overexpressing GhAP1-D3 showed significantly early flowering and early maturity without penalties for yield and fiber quality compared to wild-type(WT)plants.By contrast,the mutant lines of GhAP1-D3 generated by genome editing displayed markedly later flowering than the WT.GhAP1-D3 interacted with GhSOC1(SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1),a pivotal regulator of FTi,both in vitro and in vivo.Changes in GhAP1-D3 transcript levels clearly affected the expression of multiple key flowering regulatory genes.Additionally,DNA hypomethylation and high levels of H3K9ac affected strong expression of GhAP1-D3 in early-maturing cotton cultivars.We propose that epigenetic modifications modulate GhAP1-D3 expression to positively regulate FTi in cotton through interaction of the encoded GhAP1 with GhSOC1 and affecting the transcription of multiple flowering-related genes.These findings may also lay a foundation for breeding early-maturing cotton varieties in the future.

The transcription factor HBF1 directly activates expression of multiple flowering time repressors to delay rice flowering

Flowering time(or heading date)is an important agronomic trait that determines the environmental adaptability and yield of many crops,including rice(Oryza sativa L.).Hd3a BINDING REPRESSOR FACTOR 1(HBF1),a basic leucine zipper transcription factor,delays flowering by decreasing the expression of Early heading date 1(Ehd1),Heading date 3a(Hd3a),and RICE FLOWERING LOCUS T 1(RFT1),but the underlying molecular mechanisms have not been fully elucidated.Here,we employed the hybrid transcriptional factor(HTF)strategy to enhance the transcriptional activity of HBF1 by fusing it to four copies of the activation domain from Herpes simplex virus VP16.We discovered that transgenic rice lines overexpressing HBF1-VP64(HBF1V)show significant delays in time to flower,compared to lines overexpressing HBF1-MYC or wild-type plants,via the Ehd1–Hd3a/RFT1 pathway,under both long-day and short-day conditions.Transcriptome deep sequencing analysis indicated that 19 WRKY family genes are upregulated in the HBF1V overexpression line.We demonstrate that the previously unknown gene,OsWRKY64,is a direct downstream target of HBF1 and represses flowering in rice,whereas three known flowering repressor genes,Days to heading 7(DTH7),CONSTANS 3(OsCO3),and OsWRKY104,are also direct target genes of HBF1 in flowering regulation.Taking these results together,we propose detailed molecular mechanisms by which HBF1 regulates the time to flower in rice.

A Domestication-Associated Gene GmPRR3b Regulates the Circadian Clock and Flowering Time in Soybean

Improved soybean cultivars have been adapted to grow at a wide range of latitudes,enabling expansion of cultivation worldwide.However,the genetic basis of this broad adaptation is still not clear.Here,we report the identification of GmPRR3b as a major flowering time regulatory gene that has been selected during domestication and genetic improvement for geographic expansion.Through a genome-wide association study of a diverse soybean landrace panel consisting of 279 accessions,we identified 16 candidate quantitative loci associated with flowering time and maturity time.The strongest signal resides in the known flowering gene E2,verifying the effectiveness of our approach.We detected strong signals associated with both flowering and maturity time in a genomic region containing GmPRR3b.Haplotype analysis revealed that GmPRR3bH6 is the major form of GmPRR3b that has been utilized during recent breeding of modern cultivars.mRNA profiling analysis showed that GmPRR3bH6 displays rhythmic and photoperiod-dependent expression and is preferentially induced under long-day conditions.Overexpression of GmPRR3bH6 increased main stem node number and yield,while knockout of GmPRR3bH6 using CRISPR/Cas9 technology delayed growth and the floral transition.GmPRR3bH6 appears to act as a transcriptional repressor of multiple predicted circadian clock genes,including GmCCAIa,which directly upregulates J/GmELF3a to modulate flowering time.The causal SNP(Chr12:5520945)likely endows GmPRR3bH6 a moderate but appropriate level of activity,leading to early flowering and vigorous growth traits preferentially selected during broad adaptation of landraces and improvement of cultivars.

The Genetic Architecture of Flowering Time and Photoperiod Sensitivity in Maize as Revealed by QTL Review and Meta Analysis

The control of flowering is not only important for reproduction, but also plays a key role in the processes of domestication and adaptation. To reveal the genetic architecture for flowering time and photoperiod sensitivity, a comprehensive evaluation of the relevant literature was performed and followed by meta analysis. A total of 25 synthetic con- sensus quantitative trait loci （QTL） and four hot-spot genomic regions were identified for photoperiod sensitivity including 11 genes related to photoperiod response or flower morphogenesis and development. Besides, a comparative analysis of the QTL for flowering time and photoperiod sensitivity highlighted the regions containing shared and unique QTL for the two traits. Candidate genes associated with maize flowering were identified through integrated analysis of the homologous genes for flowering time in plants and the consensus QTL regions for photoperiod sensitivity in maize （Zea mays L.）. Our results suggest that the combination of literature review, meta-analysis and homologous blast is an efficient approach to identify new candidate genes and create a global view of the genetic architecture for maize photoperiodic flowering. Sequences of candidate genes can be used to develop molecular markers for various models of marker-assisted selection, such as marker-assisted recurrent selection and genomic selection that can contribute significantly to crop environmental adaptation.

Quantitative Trait Loci Mapping of Maize Yield and Its Components Under Different Water Treatments at Flowering Time

Drought or water stress is a serious agronomic problem resulting in maize （Zea mays L.） yield loss throughout the world. Breeding hybrids with drought tolerance is one important approach for solving this problem. However, lower efficiency and a longer period of breeding hybrids are disadvantages of traditional breeding programs. It is generally recognized that applying molecular marker techniques to traditional breeding programs could improve the efficiency of the breeding of drought-tolerant maize. To provide useful information for use in studies of maize drought tolerance, the mapping and tagging of quantitative trait loci （QTL） for yield and its components were performed in the present study on the basis of the principle of a mixed linear model. Two hundred and twenty-one recombinant inbred lines （RIL） of Yuyu 22 were grown under both well-watered and water-stressed conditions. In the former treatment group, plants were well irrigated, whereas those in the latter treatment group were stressed at flowering time. Ten plants of each genotype were grown in a row that was 3.00 m × 0.67 m （length × width）. The results show that a few of the QTL were the same （one additive QTL for ear length, two additive QTL and one pair of epistatic QTL for kernel number per row, one additive QTL for kernel weight per plant）, whereas most of other QTL were different between the two different water treatment groups. It may be that genetic expression differs under the two different water conditions. Furthermore, differences in the additive and epistatic QTL among the traits under water-stressed conditions indicate that genetic expression also differs from trait to trait. Major and minor QTL were detected for the traits, except for kernel number per row, under water-stressed conditions. Thus, the genetic mechanism of drought tolerance in maize is complex because the additive and epistatic QTL exist at the same time and the major and minor QTL all contribute to phenotype under water-stressed conditions. In particular, epidemic QTL under water-stressed conditions suggest that it is important to investigate the drought tolerance of maize from a genetic viewpoint.

The rhythmic expression of genes controlling flowering time in southern and northern populations of invasive Ambrosia artemisiifolia

Aims Flowering time has been suggested to be an important adaptive trait during the dispersal of invasive species,and identifying the molecu-lar mechanisms underlying flowering time may provide insight into the local adaptation during the process of invasion.Here,we con-ducted a preliminary exploration on the genetic basis of the differ-entiation of flowering time in Ambrosia artemisiifolia.Methods using relative real-time fluorescent quantitative polymerase chain reaction,we investigated the expression levels of eight flowering-related genes,including AP1,FT,SOC1,CRY2,FKF1,GI,CO2 and SPY,in leaves and flowers at different time points in individuals from northern beijing and southern Wuhan populations that exhibit significant differences in flowering times to identify any rhythmic changes in gene expression and their association with differential flowering times.Important Findings The differentiation of flowering time in the A.artemisiifolia popula-tions was closely associated with five genes involved in flowering pathways.The floral pathway integrators FT and SOC1 and floral meristem identity gene AP1 exhibited increased expression during flowering.The photoreceptor CRY2 in the light-dependent path-way and the SPY gene in the gibberellin pathway displayed specific expression patterns over time.in earlier-flowering beijing plants,CRY2 expression was lower and SPY expression was higher than in Wuhan plants.The expression patterns of these five genes sug-gest a molecular basis for the differentiation of flowering time in A.artemisiifolia.

Arabidopsis PWWP domain proteins mediate H_3K_(27) trimethylation on FLC and regulate flowering time

LHP1 mediates recruitment of the PRC2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H3K27me3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain proteins （PDPs） interact with FVE and MSI5 to suppress FLC expression and thereby promote flowering. We demonstrated that FVE, MSI5, and PDP3 were co-purified with LHP1. The H3K27me3 level on FLC was decreased in the pdp mutants as welt as in the fve/ msi5 double mutant. This study suggests that PDPs function together with FVE and MSI5 to regulate the function of the PRC2 complex on FLC.

Divergence in flowering time is a major component contributing to reproductive isolation between two wild rice species(Oryza rufipogon and O. nivara)

It is of critical importance for our understanding of speciation process to determine the forms of reproductive isolation and their relative importance in species divergence. Oryza nivara and O. rufipogon are direct ancestors of Asian cultivated rice and a progenitor-daughter species pair. Investigating the reproductive isolation between them provides insights into plant speciation and helps understanding of the rice domestication. Here, we quantitatively measured the major components of reproductive isolation between the two species based on common garden and crossing experiments for three pairs of sympatric populations in Nepal, Cambodia and Laos.We revealed significant differences in the flowering times between species pairs, with O. nivara flowering much earlier than O. rufipogon. A very weak reduction in seed set but no reduction in F1 viability and fertility were detected for the crosses between species relative to those within species. Moreover, we detected asymmetrical compatibility between species and found that emasculation significantly decreased pollination success in O. nivara but not in O. rufipogon.Our study demonstrates that the divergence between O. nivara and O. rufipogon is maintained almost entirely by the difference in flowering times and suggests that differential flowering times contribute to both habitat preferences and reproductive isolation between species.