Photoperiodism dynamics during the domestication and improvement of soybean

Soybean (Glycine max) is a facultative short-day plant with a sensitive photoperiod perception and reaction system, which allows it to adjust its physiological state and gene regulatory networks to seasonal and diurnal changes in environmental conditions. In the past few decades, soybean cultivation has spread from East Asia to areas throughout the world. Biologists and breeders must now confront the challenge of understanding the molecular mechanism of soybean photoperiodism and improving agronomic traits to enable this important crop to adapt to geographical and environmental changes. In this review, we summarize the genetic regulatory network underlying photoperiodic responses in soybean. Genomic and genetic studies have revealed that the circadian clock, in conjunction with the light perception pathways, regulates photoperiodic flowering. Here, we provide an annotated list of 844 candidate flowering genes in soybean, with their putative biological functions. Many photoperiod-related genes have been intensively selected during domestication and crop improvement. Finally, we describe recent progress in engineering photoperiod-responsive genes for improving agronomic traits to enhance geographic adaptation in soybean, as well as future prospects for research on soybean photoperiodic responses.     

Comparative genomic analysis of soybean flowering genes

Flowering is an important agronomic trait that determines crop yield. Soybean is a major oilseed legume crop used for human and animal feed. Legumes have unique vegetative and floral complexities. Our understanding of the molecular basis of flower initiation and development in legumes is limited. Here, we address this by using a computational approach to examine flowering regulatory genes in the soybean genome in comparison to the most studied model plant, Arabidopsis. For this comparison, a genome-wide analysis of orthologue groups was performed, followed by an in silico gene expression analysis of the identified soybean flowering genes. Phylogenetic analyses of the gene families highlighted the evolutionary relationships among these candidates. Our study identified key flowering genes in soybean and indicates that the vernalisation and the ambient-temperature pathways seem to be the most variant in soybean. A comparison of the orthologue groups containing flowering genes indicated that, on average, each Arabidopsis flowering gene has 2-3 orthologous copies in soybean. Our analysis highlighted that the CDF3, VRN1, SVP, AP3 and PIF3 genes are paralogue-rich genes in soybean. Furthermore, the genome mapping of the soybean flowering genes showed that these genes are scattered randomly across the genome. A paralogue comparison indicated that the soybean genes comprising the largest orthologue group are clustered in a 1.4 Mb region on chromosome 16 of soybean. Furthermore, a comparison with the undomesticated soybean (Glycine soja) revealed that there are hundreds of SNPs that are associated with putative soybean flowering genes and that there are structural variants that may affect the genes of the light-signalling and ambient-temperature pathways in soybean. Our study provides a framework for the soybean flowering pathway and insights into the relationship and evolution of flowering genes between a short-day soybean and the long-day plant, Arabidopsis.     

The quantitative response of wheat vernalization to environmental variables indicates that vernalization is not a response to cold temperature

The initiation of flowering is a crucial trait that allows temperate plants to flower in the favourable conditions of spring. The timing of flowering initiation is governed by two main mechanisms: vernalization that defines a plant's requirement for a prolonged exposure to cold temperatures; and photoperiod sensitivity defining the need for long days to initiate floral transition. Genetic variability in both vernalization and photoperiod sensitivity largely explains the adaptability of cultivated crop plants such as bread wheat (Triticum aestivum L.) to a wide range of climatic conditions. The major genes controlling wheat vernalization (VRN1, VRN2, and VRN3) and photoperiod sensitivity (PPD1) have been identified, and knowledge of their interactions at the molecular level is growing. However, the quantitative effects of temperature and photoperiod on these genes remain poorly understood. Here it is shown that the distinction between the temperature effects on organ appearance rate and on vernalization sensu stricto is crucial for understanding the quantitative effects of the environmental signal on wheat flowering. By submitting near isogenic lines of wheat differing in their allelic composition at the VRN1 locus to various temperature and photoperiod treatments, it is shown that, at the whole-plant level, the vernalization process has a positive response to temperature with complex interactions with photoperiod. In addition, the phenotypic variation associated with the presence of different spring homoeoalleles of VRN1 is not induced by a residual vernalization requirement. The results demonstrate that a precise definition of vernalization is necessary to understand and model temperature and photoperiod effects on wheat flowering. It is suggested that this definition should be used as the basis for gene expression studies and assessment of functioning of the wheat flowering gene network, including an explicit account of the quantitative effect of environmental variables.     

Rapid evolution of the plastid translational apparatus in a nonphotosynthetic plant: Loss or accelerated sequence evolution of tRNA and ribosomal protein genes

Summary The vestigial plastid genome of Epifagus virginiana (beechdrops), a nonphotosynthetic parasitic flowering plant, is functional but lacks six ribosomal protein and 13 tRNA genes found in the chloroplast DNAs of photosynthetic flowering plants. Import of nuclear gene products is hypothesized to compensate for many of these losses. Codon usage and amino acid usage patterns in Epifagus plastic genes have not been affected by the tRNA gene losses, though a small shift in the base composition of the whole genome (toward A + T -richness) is apparent. The ribosomal protein and tRNA genes that remain have had a high rate of molecular evolution, perhaps due to relaxation of constraints on the translational apparatus. Despite the compactness and extensive gene loss, one translational gene (infA, encoding initiation factor 1) that is a pseudogene in tobacco has been maintained intact in Epifagus.Offprint requests to: J.D. Palmer     

The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.     

Expression of flowering-time genes in soybean E1 near-isogenic lines under short and long day conditions

Control of soybean flowering time is important for geographic adaptation and maximizing yield. Plant breeders have identified a series of genes (E genes) that condition time to flowering; however, the molecular basis in the control of flowering by these E genes, in conjunction with canonical flowering-time genes, has not been studied. Time to flowering in near-isogenic lines (NILs) at the E1 locus was tested using a reciprocal transfer experiment under short day (SD) and long day (LD) conditions. Beginning 8 days after planting, three plant samples were harvested every 3 h for a 48-h period. RNA was isolated from these plants, and RNA samples were pooled for each line and each time period for cDNA synthesis. RT-PCR analysis was performed using primers synthesized for a number of putative flowering-time genes based on homology of soybean EST and genomic sequences to Arabidopsis genes. The results of the reciprocal transfer experiment suggest that the pre-inductive photoperiod-sensitive phase of the E1 NILs responsible for inducing flowering is perceived as early as 5–7-day post-planting. No gene expression differences were found between the E1 and e1 NILs, suggesting that the E1 gene does not directly affect the flowering-time genes during the time period tested; however, differences were observed in gene expression between SD and LD treatments for the putative soybean TOC1, CO, and FT genes. The gene expression results in this study were similar to those of flowering-time genes found in other SD species, suggesting that the selected genes correspond to the soybean flowering-time orthologs.     

Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean

The genomes of most, if not all, flowering plants have undergone whole genome duplication events during their evolution. The impact of such polyploidy events is poorly understood, as is the fate of most duplicated genes. We sequenced an approximately 1 million-bp region in soybean (Glycine max) centered on the Rpg1-b disease resistance gene and compared this region with a region duplicated 10 to 14 million years ago. These two regions were also compared with homologous regions in several related legume species (a second soybean genotype, Glycine tomentella, Phaseolus vulgaris, and Medicago truncatula), which enabled us to determine how each of the duplicated regions (homoeologues) in soybean has changed following polyploidy. The biggest change was in retroelement content, with homoeologue 2 having expanded to 3-fold the size of homoeologue 1. Despite this accumulation of retroelements, over 77% of the duplicated low-copy genes have been retained in the same order and appear to be functional. This finding contrasts with recent analyses of the maize (Zea mays) genome, in which only about one-third of duplicated genes appear to have been retained over a similar time period. Fluorescent in situ hybridization revealed that the homoeologue 2 region is located very near a centromere. Thus, pericentromeric localization, per se, does not result in a high rate of gene inactivation, despite greatly accelerated retrotransposon accumulation. In contrast to low-copy genes, nucleotide-binding-leucine-rich repeat disease resistance gene clusters have undergone dramatic species/homoeologue-specific duplications and losses, with some evidence for partitioning of subfamilies between homoeologues.     

On the potential strength and consequences for nonrandom gene flow caused by local adaptation in flowering time

Gene flow is generally considered a random process, that is the loci under consideration have no effect on dispersal success. Edelaar and Bolnick (Trends Ecol Evol, 27, 2012 659) recently argued that nonrandom gene flow could exert a significant evolutionary force. It can, for instance, ameliorate the maladaptive effects of immigration into locally adapted populations. I examined the potential strength for nonrandom gene flow for flowering time genes, a trait frequently found to be locally adapted. The idea is that plants that successfully export pollen into a locally adapted resident population will be a genetically biased subset of their natal population – they will have resident‐like flowering times. Reciprocally, recipients will be more migrant‐like than the resident population average. I quantified the potential for biased pollen exchange among three populations along a flowering time cline in Brassica rapa from southern California. A two‐generation line cross experiment demonstrated genetic variance in flowering time, both within and among populations. Calculations based on the variation in individual flowering schedules showed that resident plants with the most migrant‐like flowering times could expect to have up to 10 times more of the their flowers pollinated by immigrant pollen than the least migrant‐like. Further, the mean flowering time of the pollen exporters that have access to resident mates differs by up to 4 weeks from the mean in the exporters’ natal population. The data from these three populations suggest that the bias in gene flow for flowering time cuts the impact on the resident population by as much as half. This implies that when selection is divergent between populations, migrants with the highest mating success tend to be resident‐like in their flowering times, and so, fewer maladaptive alleles will be introduced into the locally adapting gene pool.     

The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.     

Molecular mechanisms for the photoperiodic regulation of flowering in soybean

Photoperiodic flowering is one of the most important factors affecting regional adaptation and yield in soybean (Glycine max). Plant adaptation to long-day conditions at higher latitudes requires early flowering and a reduction or loss of photoperiod sensitivity; adaptation to short-day conditions at lower latitudes involves delayed flowering, which prolongs vegetative growth for maximum yield potential. Due to the influence of numerous major loci and quantitative trait loci (QTLs), soybean has broad adaptability across latitudes. Forward genetic approaches have uncovered the molecular basis for several of these major maturity genes and QTLs. Moreover, the molecular characterization of orthologs of Arabidopsis thaliana flowering genes has enriched our understanding of the photoperiodic flowering pathway in soybean. Building on early insights into the importance of the photoreceptor phytochrome A, several circadian clock components have been integrated into the genetic network controlling flowering in soybean: E1, a repressor of FLOWERING LOCUS T orthologs, plays a central role in this network. Here, we provide an overview of recent progress in elucidating photoperiodic flowering in soybean, how it contributes to our fundamental understanding of flowering time control, and how this information could be used for molecular design and breeding of high-yielding soybean cultivars.     

Identification of Heterodera glycines (soybean cyst nematode [SCN]) cDNA sequences with high identity to those of Caenorhabditis elegans having lethal mutant or RNAi phenotypes

The soybean cyst nematode (SCN; Heterodera glycines) is a devastating obligate parasite of Glycine max (soybean) causing one billion dollars in losses to the US economy per year and over ten billion dollars in losses worldwide. While much is understood about the pathology of H. glycines, its genome sequence is not well characterized or fully sequenced. We sought to create bioinformatic tools to mine the H. glycines nucleotide database. One way is to use a comparative genomics approach by anchoring our analysis with an organism, like the free-living nematode Caenorhabditis elegans. Unlike H. glycines, the C. elegans genome is fully sequenced and is well characterized with a number of lethal genes identified through experimental methods. We compared an EST database of H. glycines with the C. elegans genome. Our goal was identifying genes that may be essential for H. glycines survival and would serve as an automated pipeline for RNAi studies to both study and control H. glycines. Our analysis yielded a total of nearly 8334 conserved genes between H. glycines and C. elegans. Of these, 1508 have lethal phenotypes/phenocopies in C. elegans. RNAi of a conserved ribosomal gene from H. glycines (Hg-rps-23) yielded dead and dying worms as shown by positive Sytox fluorescence. Endogenous Hg-rps-23 exhibited typical RNA silencing as shown by RT-PCR. However, an unrelated gene Hg-unc-87 did not exhibit RNA silencing in the Hg-rps-23 dsRNA-treated worms, demonstrating the specificity of the silencing.     

大豆向热带地区发展的遗传基础

大豆(Glycine max)是光周期敏感的植物,该特性是决定其生育期及其生态适应区的关键因素。温带的大豆品种引种到热带地区(短日照)时,开花期和成熟期提前、产量降低,限制了大豆在热带地区的种植。长童期(LJ)大豆品种的发现是解决该问题的重要突破。在短日照条件下,LJ品种比温带品种开花晚、体量大、成熟晚且产量提高。前期研究发现,J位点是控制LJ性状的关键位点。近期,我国科学家通过精细定位克隆了J基因,发现其与拟南芥(Arabidopsis thaliana)早花基因(ELF3)同源。他们通过功能互补和近等基因系等方法验证了J基因的功能,在短日照条件下,等位基因j比J开花晚、成熟晚且产量提高。进一步研究发现,J蛋白与E1基因(豆科植物开花抑制因子)的启动子结合抑制E1基因的表达,从而解除E1对大豆开花基因(FT)的抑制,促进大豆在短日照下开花。研究还发现在大豆种质资源中存在多种j等位变异。该研究引领了大豆生育期遗传研究的新方向,揭示了大豆向热带地区发展的遗传基础。     

Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b,an ortholog of FLOWERING LOCUS T

Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a ‘weight’ model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.     

Analysis of clock gene homologs using unifoliolates as target organs in soybean (Glycine max)

Soybean is a typical short-day crop, and its photoperiodic response of flowering is critical to its yield. This phenomenon was well studied during the last century, but the molecular mechanism governing it is unknown. The clock-gene homologs, GmLCL1 and GmLCL2 (Glycine max LHY/CCA1 Like 1 and 2) and GmTOC1 (Glycine max TOC1) were cloned from Glycine max L. KN18, and their expression patterns were analyzed using a system developed in this study. We employed 8h light/16h dark and 18h light/6h dark as short- and long-day conditions, respectively, because in these conditions soybean plants had significant indexes of flowering time. We also used unifoliolates, not the whole plant as Arabidopsis, as target organs for gene expression analysis. GmLCL1 and GmLCL2 had similar circadian expression patterns and both were morning genes, while GmTOC1 was an evening gene and peaked in the evening. The expressions of GmLCL1 and GmLCL2 were obviously antiphase to that of GmTOC1.Our study provided a system that simplified the experiments without compromising the quality of data obtained and was suitable for analyzing the molecular mechanism of flowering in soybean. GmLCL1, GmLCL2 and GmTOC1 may be the components of central clock in soybean.     

Rapid birth-death evolution specific to xenobiotic cytochrome P450 genes in vertebrates

Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50-80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth-death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.     

Conservation of Arabidopsis flowering genes in model legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.