Physical seed dormancy in pea is genetically separable from seed coat thickness and roughness.

Introduction: The seeds of wild pea (Pisum) exhibit marked physical dormancy due to impermeability of the seed coat to water, and the loss of this dormancy is thought to have been critical for domestication. Wild pea seed coats are also notably thick and rough, traits that have also reduced during domestication and are anecdotally linked to increased permeability. However, how these traits specifically interact with permeability is unclear. Methods: To investigate this, we examined the genetic control of differences in seed coat characteristics between wild P. sativum ssp. humile and a non-dormant domesticated P. s. sativum accession in a recombinant inbred population. QTL effects were confirmed and their locations refined in segregating F4/5 populations. Results: In this population we found a moderate correlation between testa thickness and permeability, and identified loci that affect them independently, suggesting no close functional association. However, the major loci affecting both testa thickness and permeability collocated closely with Mendel's pigmentation locus A, suggesting flavonoid compounds under its control might contribute significantly to both traits. We also show that seed coat roughness is oligogenic in this population, with the major locus independent of both testa thickness and permeability, suggesting selection for smooth seed was unlikely to be due to effects on either of these traits. Discussion: Results indicate loss of seed coat dormancy during domestication was not primarily driven by reduced testa thickness or smooth seededness. The close association between major permeability and thickness QTL and Mendel's 'A' warrant further study, particularly regarding the role of flavonoids.

Focusing the GWAS Lens on days to flower using latent variable phenotypes derived from global multienvironment trials.

Adaptation constraints within crop species have resulted in limited genetic diversity in some breeding programs and areas where new crops have been introduced, for example, for lentil (Lens culinaris Medik.) in North America. An improved understanding of the underlying genetics involved in phenology-related traits is valuable knowledge to aid breeders in overcoming limitations associated with unadapted germplasm and expanding their genetic diversity by introducing new, exotic material. We used a large, 18 site-year, multienvironment dataset phenotyped for phenology-related traits across nine locations and over 3 yr along with accompanying latent variable phenotypes derived from a photothermal model and principal component analysis (PCA) of days from sowing to flower (DTF) data for a lentil diversity panel (324 accessions), which has also been genotyped with an exome capture array. Genome-wide association studies (GWAS) on DTF across multiple environments helped confirm associations with known flowering-time genes and identify new quantitative trait loci (QTL), which may contain previously unknown flowering time genes. Additionally, the use of latent variable phenotypes, which can incorporate environmental data such as temperature and photoperiod as both GWAS traits and as covariates, strengthened associations, revealed additional hidden associations, and alluded to potential roles of the associated QTL. Our approach can be replicated with other crop species, and the results from our GWAS serve as a resource for further exploration into the complex nature of phenology-related traits across the major growing environments for cultivated lentil.

Identification and characterization of putative targets of VEGETATIVE1/FULc, a key regulator of development of the compound inflorescence in pea and related legumes.

Inflorescence architecture contributes to essential plant traits. It determines plant shape, contributing to morphological diversity, and also determines the position and number of flowers and fruits produced by the plant, thus influencing seed yield. Most legumes have compound inflorescences, where flowers are produced in secondary inflorescences (I2), formed at the flanks of the main primary inflorescence (I1), in contrast to simple inflorescences of plants like Arabidopsis, in which flowers are directly formed on the I1. The pea VEGETATIVE1/FULc (VEG1) gene, and its homologs in other legumes, specify the formation of the I2 meristem, a function apparently restricted to legumes. To understand the control of I2 development, it is important to identify the genes working downstream of VEG1. In this study, we adopted a novel strategy to identify genes expressed in the I2 meristem, as potential regulatory targets of VEG1. To identify pea I2-meristem genes, we compared the transcriptomes of inflorescence apices from wild-type and mutants affected in I2 development, such as proliferating inflorescence meristems (pim, with more I2 meristems), and veg1 and vegetative2 (both without I2 meristems). Analysis of the differentially expressed genes using Arabidopsis genome databases combined with RT-qPCR expression analysis in pea allowed the selection of genes expressed in the pea inflorescence apex. In situ hybridization of four of these genes showed that all four genes are expressed in the I2 meristem, proving our approach to identify I2-meristem genes was successful. Finally, analysis by VIGS (virus-induced gene silencing) in pea identified one gene, PsDAO1, whose silencing leads to small plants, and another gene, PsHUP54, whose silencing leads to plants with very large stubs, meaning that this gene controls the activity of the I2 meristem. PsHUP54-VIGS plants are also large and, more importantly, produce large pods with almost double the seeds as the control. Our study shows a new useful strategy to isolate I2-meristem genes and identifies a novel gene, PsHUP54, which seems to be a promising tool to improve yield in pea and in other legumes.

Genetic control of the operculum and capsule morphology of Eucalyptus globulus.

BACKGROUND AND AIMS: The petaline operculum that covers the inner whorls until anthesis and the woody capsule that develops after fertilization are reproductive structures of eucalypts that protect the flower and seeds. Although they are distinct organs, they both develop from flower buds and this common ontogeny suggests shared genetic control. In Eucalyptus globulus their morphology is variable and we aimed to identify the quantitative trait loci (QTL) underlying this variation and determine whether there is common genetic control of these ecologically and taxonomically important reproductive structures. METHODS: Samples of opercula and capsules were collected from 206 trees that belong to a large outcrossed F2E. globulus mapping population. The morphological variation in these structures was characterized by measuring six operculum and five capsule traits. QTL analysis was performed using these data and a linkage map consisting of 480 markers. KEY RESULTS: A total of 27 QTL were detected for operculum traits and 28 for capsule traits, with the logarithm of odds ranging from 2.8 to 11.8. There were many co-located QTL associated with operculum or capsule traits, generally reflecting allometric relationships. A key finding was five genomic regions where co-located QTL affected both operculum and capsule morphology, and the overall trend for these QTL was to affect elongation of both organs. Some of these QTL appear to have a significant effect on the phenotype, with the strongest QTL explaining 26.4 % of the variation in operculum shape and 16.4 % in capsule shape. Flower bud measurements suggest the expression of these QTL starts during bud development. Several candidate genes were found associated with the QTL and their putative function is discussed. CONCLUSIONS: Variation in both operculum and capsule traits in E. globulus is under strong genetic control. Our results suggest that these reproductive structures share a common genetic pathway during flower bud development.

The genetic architecture of flowering time changes in pea from wild to crop.

Change in phenology has been an important component in crop evolution, and selection for earlier flowering through a reduction in environmental sensitivity has helped broaden adaptation in many species. Natural variation for flowering in domesticated pea (Pisum sativum L.) has been noted and studied for decades, but there has been no clear account of change relative to its wild progenitor. Here we examined the genetic control of differences in flowering time between wild P. sativum ssp. humile and a typical late-flowering photoperiodic P. s. sativum accession in a recombinant inbred population under long and short photoperiods. Our results confirm the importance of the major photoperiod sensitivity locus Hr/PsELF3a and identify two other loci on chromosomes 1 (DTF1) and 3 (DTF3) that contribute to earlier flowering in the domesticated line under both photoperiods. The domesticated allele at a fourth locus on chromosome 6 (DTF6) delays flowering under long days only. Map positions, inheritance patterns, and expression analyses in near-isogenic comparisons imply that DTF1, DTF3, and DTF6 represent gain-of-function alleles of the florigen/antiflorigen genes FTa3, FTa1, and TFL1c/LF, respectively. This echoes similar variation in chickpea and lentil, and suggests a conserved route to reduced photoperiod sensitivity and early phenology in temperate pulses.

Genetic analysis of early phenology in lentil identifies distinct loci controlling component traits.

Modern-day domesticated lentil germplasm is generally considered to form three broad adaptation groups: Mediterranean, South Asian, and northern temperate, which correspond to the major global production environments. Reproductive phenology plays a key role in lentil adaptation to this diverse ecogeographic variation. Here, we dissect the characteristic earliness of the pilosae ecotype, suited to the typically short cropping season of South Asian environments. We identified two loci, DTF6a and DTF6b, at which dominant alleles confer early flowering, and we show that DTF6a alone is sufficient to confer early flowering under extremely short photoperiods. Genomic synteny confirmed the presence of a conserved cluster of three florigen (FT) gene orthologues among potential candidate genes, and expression analysis in near-isogenic material showed that the early allele is associated with a strong derepression of the FTa1 gene in particular. Sequence analysis revealed a 7.4 kb deletion in the FTa1-FTa2 intergenic region in the pilosae parent, and a wide survey of >350 accessions with diverse origin showed that the dtf6a allele is predominant in South Asian material. Collectively, these results contribute to understanding the molecular basis of global adaptation in lentil, and further emphasize the importance of this conserved genomic region for adaptation in temperate legumes generally.

A Point Mutation in Phytochromobilin synthase Alters the Circadian Clock and Photoperiodic Flowering of Medicago truncatula.

Plants use seasonal cues to initiate flowering at an appropriate time of year to ensure optimal reproductive success. The circadian clock integrates these daily and seasonal cues with internal cues to initiate flowering. The molecular pathways that control the sensitivity of flowering to photoperiods (daylengths) are well described in the model plant Arabidopsis. However, much less is known for crop species, such as legumes. Here, we performed a flowering time screen of a TILLING population of Medicago truncatula and found a line with late-flowering and altered light-sensing phenotypes. Using RNA sequencing, we identified a nonsense mutation in the Phytochromobilin synthase (MtPΦBS) gene, which encodes an enzyme that carries out the final step in the biosynthesis of the chromophore required for phytochrome (phy) activity. The analysis of the circadian clock in the MtpΦbs mutant revealed a shorter circadian period, which was shared with the MtphyA mutant. The MtpΦbs and MtphyA mutants showed downregulation of the FT floral regulators MtFTa1 and MtFTb1/b2 and a change in phase for morning and night core clock genes. Our findings show that phyA is necessary to synchronize the circadian clock and integration of light signalling to precisely control the timing of flowering.

Genetic basis for lentil adaptation to summer cropping in northern temperate environments.

The continued success of lentil (Lens culinaris Medik.) genetic improvement relies on the availability of broad genetic diversity, and new alleles need to be identified and incorporated into the cultivated gene pool. Availability of robust and predictive markers greatly enhances the precise transfer of genomic regions from unadapted germplasm. Quantitative trait loci (QTL) for key phenological traits in lentil were located using a recombinant inbreed line (RIL) population derived from a cross between an Ethiopian landrace (ILL 1704) and a northern temperate cultivar (CDC Robin). Field experiments were conducted at Sutherland research farm in Saskatoon and at Rosthern, Saskatchewan, Canada during 2018 and 2019. A linkage map was constructed using 21,634 single nucleotide polymorphisms (SNPs) located on seven linkage groups (LGs), which correspond to the seven haploid chromosomes of lentil. Eight QTL were identified for six phenological traits. Flowering-related QTL were identified at two regions on LG6. FLOWERING LOCUS T (FT) genes were annotated within the flowering time QTL interval based on the lentil reference genome. Similarly, a major QTL for postflowering developmental processes was located on LG5 with several senescence-associated genes annotated within the QTL interval. The flowering time QTL was validated in a different genetic background indicating the potential use of the identified markers for marker-assisted selection to precisely transfer genomic regions from exotic germplasm into elite crop cultivars without disrupting adaptation.

Genetic basis and adaptation trajectory of soybean from its temperate origin to tropics.

Soybean (Glycine max) serves as a major source of protein and edible oils worldwide. The genetic and genomic bases of the adaptation of soybean to tropical regions remain largely unclear. Here, we identify the novel locus Time of Flowering 16 (Tof16), which confers delay flowering and improve yield at low latitudes and determines that it harbors the soybean homolog of LATE ELONGATED HYPOCOTYL (LHY). Tof16 and the previously identified J locus genetically additively but independently control yield under short-day conditions. More than 80% accessions in low latitude harbor the mutations of tof16 and j, which suggests that loss of functions of Tof16 and J are the major genetic basis of soybean adaptation into tropics. We suggest that maturity and yield traits can be quantitatively improved by modulating the genetic complexity of various alleles of the LHY homologs, J and E1. Our findings uncover the adaptation trajectory of soybean from its temperate origin to the tropics.

Overcoming the genetic compensation response of soybean florigens to improve adaptation and yield at low latitudes.

The classical soybean (Glycine max) trait long juvenile (LJ) is essentially a reduction in sensitivity to short-day (SD) conditions for induction and completion of flowering, and has been introduced into soybean cultivars to improve yield in tropical environments. However, only one locus, J, is known to confer LJ in low-latitude varieties. Here, we defined two quantitative trait loci contributing to the LJ trait, LJ16.1 and LJ16.2, and identified them as the florigen (FT) homologs FT2a and FT5a, respectively. The two selected florigen variations both delay flowering time under SD conditions by repressing the floral meristem identity gene GmAPETALA1. Single mutants have a relatively subtle effect on flowering time and displayed a substantial genetic compensation response, but this was absent in ft2a ft5a double mutants, which showed an enhanced LJ phenotype that translated to higher yields under SD conditions. A survey of sequence diversity suggests that FT2a and FT5a variants have diverse origins and have played distinct roles as soybean spread to lower latitudes. Our results show that integration of variants in the florigen genes offers a strategy for customizing flowering time to adjust adaptation and improve crop productivity in tropical regions.

Genetic and gene expression analysis of flowering time regulation by light quality in lentil.

BACKGROUND AND AIMS: Flowering time is important due to its roles in plant adaptation to different environments and subsequent formation of crop yield. Changes in light quality affect a range of developmental processes including flowering time, but little is known about light quality-induced flowering time control in lentil. This study aims to investigate the genetic basis for differences in flowering response to light quality in lentil. METHODS: We explored variation in flowering time caused by changes in red/far-red-related light quality environments of a lentil interspecific recombinant inbred line (RIL) population developed from a cross between Lens culinaris cv. Lupa and L. orientalis accession BGE 016880. A genetic linkage map was constructed and then used for identifying quantitative trait loci (QTLs) associated with flowering time regulation under different light quality environments. Differential gene expression analysis through transcriptomic study and RT-qPCR were used to identify potential candidate genes. KEY RESULTS: QTL mapping located 13 QTLs controlling flower time under different light quality environments, with phenotypic variance explained ranging from 1.7 to 62.9 %. Transcriptomic profiling and gene expression analysis for both parents of this interspecific RIL population identified flowering-related genes showing environment-specific differential expression (flowering DEGs). One of these, a member of the florigen gene family FTa1 (LcFTa1), was located close to three major QTLs. Furthermore, gene expression results suggested that two other florigen genes (LcFTb1 and LcFTb2), MADS-box transcription factors such as LcAGL6/13d, LcSVPb, LcSOC1b and LcFULb, as well as bHLH transcription factor LcPIF6 and Gibberellin 20 oxidase LcGA20oxC,G may also be involved in the light quality response. CONCLUSIONS: Our results show that a major component of flowering time sensitivity to light quality is tightly linked to LcFTa1 and associated with changes in its expression. This work provides a foundation for crop improvement of lentil with better adaptation to variable light environments.

Ancient relaxation of an obligate short-day requirement in common bean through loss of CONSTANS-like gene function.

Common bean (Phaseolus vulgaris L.) is a major global food staple and source of dietary protein that was domesticated independently in Mexico and Andean South America. Its subsequent development as a crop of importance worldwide has been enabled by genetic relaxation of the strict short-day requirement typical of wild forms, but the genetic basis for this change is not well understood. Recently, a loss of photoperiod sensitivity was shown to result from mutations in the phytochrome photoreceptor gene Ppd/PHYA3 that arose independently within the two major domesticated lineages. Here, we define a second major photoperiod sensitivity locus, at which recessive alleles associate with deleterious mutations affecting the CONSTANS-like gene COL2. A wider survey of sequence variation in over 800 diverse lines, including wild, landrace, and domesticated accessions, show that distinct col2 haplotypes are associated with early flowering in Andean and Mesoamerican germplasm. The relative frequencies and distributions of COL2 and PHYA3 haplotypes imply that photoperiod adaptation developed in two phases within each gene pool: an initial reduction in sensitivity through impairment of COL2 function and subsequent complete loss through PHYA3. Gene expression analyses indicate that COL2 functions downstream of PHYA3 to repress expression of FT genes and may function in parallel with PvE1, the bean ortholog of a key legume-specific flowering repressor. Collectively, these results define the molecular basis for a key phenological adaptation, reveal a striking convergence in the naturally replicated evolution of this major crop, and further emphasize the wider evolutionary lability of CONSTANS effects on flowering time control.

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation.

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

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 bZIP transcription factor FDc1 physically interacts with Dt1, and the FDc1-Dt1 complex directly represses the expression of APETALA1 (AP1). We propose that FT5a 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 FT5a in controlling the stem growth habit and flowering time in soybean, which determine the adaptability and grain yield of this important crop.

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.

Stepwise selection on homeologous PRR genes controlling flowering and maturity during soybean domestication.

Adaptive changes in plant phenology are often considered to be a feature of the so-called 'domestication syndrome' that distinguishes modern crops from their wild progenitors, but little detailed evidence supports this idea. In soybean, a major legume crop, flowering time variation is well characterized within domesticated germplasm and is critical for modern production, but its importance during domestication is unclear. Here, we identify sequential contributions of two homeologous pseudo-response-regulator genes, Tof12 and Tof11, to ancient flowering time adaptation, and demonstrate that they act via LHY homologs to promote expression of the legume-specific E1 gene and delay flowering under long photoperiods. We show that Tof12-dependent acceleration of maturity accompanied a reduction in dormancy and seed dispersal during soybean domestication, possibly predisposing the incipient crop to latitudinal expansion. Better understanding of this early phase of crop evolution will help to identify functional variation lost during domestication and exploit its potential for future crop improvement.

The CYCLIN-DEPENDENT KINASE Module of the Mediator Complex Promotes Flowering and Reproductive Development in Pea.

Control of flowering time has been a major focus of comparative genetic analyses in plant development. This study reports on a forward genetic approach to define previously uncharacterized components of flowering control pathways in the long-day legume, pea (Pisum sativum). We isolated two complementation groups of late-flowering mutants in pea that define two uncharacterized loci, LATE BLOOMER3 (LATE3) and LATE4, and describe their diverse effects on vegetative and reproductive development. A map-based comparative approach was employed to identify the underlying genes for both loci, revealing that that LATE3 and LATE4 are orthologs of CYCLIN DEPENDENT KINASE8 (CDK8) and CYCLIN C1 (CYCC1), components of the CDK8 kinase module of the Mediator complex, which is a deeply conserved regulator of transcription in eukaryotes. We confirm the genetic and physical interaction of LATE3 and LATE4 and show that they contribute to the transcriptional regulation of key flowering genes, including the induction of the florigen gene FTa1 and repression of the floral repressor LF Our results establish the conserved importance of the CDK8 module in plants and provide evidence for the function of CYCLIN C1 orthologs in the promotion of flowering and the maintenance of normal reproductive development.

Altered Expression of an FT Cluster Underlies a Major Locus Controlling Domestication-Related Changes to Chickpea Phenology and Growth Habit.

Flowering time is a key trait in breeding and crop evolution, due to its importance for adaptation to different environments and for yield. In the particular case of chickpea, selection for early phenology was essential for the successful transition of this species from a winter to a summer crop. Here, we used genetic and expression analyses in two different inbred populations to examine the genetic control of domestication-related differences in flowering time and growth habit between domesticated chickpea and its wild progenitor Cicer reticulatum. A single major quantitative trait locus for flowering time under short-day conditions [Days To Flower (DTF)3A] was mapped to a 59-gene interval on chromosome three containing a cluster of three FT genes, which collectively showed upregulated expression in domesticated relative to wild parent lines. An equally strong association with growth habit suggests a pleiotropic effect of the region on both traits. These results indicate the likely molecular explanation for the characteristic early flowering of domesticated chickpea, and the previously described growth habit locus Hg. More generally, they point to de-repression of this specific gene cluster as a conserved mechanism for achieving adaptive early phenology in temperate legumes.

Parallel origins of photoperiod adaptation following dual domestications of common bean.

Common bean (Phaseolus vulgaris L.) is an important grain legume domesticated independently in Mexico and Andean South America approximately 8000 years ago. Wild forms are obligate short-day plants, and relaxation of photoperiod sensitivity was important for expansion to higher latitudes and subsequent global spread. To better understand the nature and origin of this key adaptation, we examined its genetic control in progeny of a wide cross between a wild accession and a photoperiod-insensitive cultivar. We found that photoperiod sensitivity is under oligogenic control, and confirm a major effect of the Ppd locus on chromosome 1. The red/far-red photoreceptor gene PHYTOCHROME A3 (PHYA3) was identified as a strong positional candidate for Ppd, and sequencing revealed distinct deleterious PHYA3 mutations in photoperiod-insensitive Andean and Mesoamerican accessions. These results reveal the independent origins of photoperiod insensitivity within the two major common bean gene pools and demonstrate the conserved importance of PHYA genes in photoperiod adaptation of short-day legume species.