Sainfoin (Onobrychis spp.) crop ontology: supporting germplasm characterization and international research collaborations.

Sainfoin (Onobrychis spp.) is a perennial forage legume that is also attracting attention as a perennial pulse with potential for human consumption. The dual use of sainfoin underpins diverse research and breeding programs focused on improving sainfoin lines for forage and pulses, which is driving the generation of complex datasets describing high dimensional phenotypes in the post-omics era. To ensure that multiple user groups, for example, breeders selecting for forage and those selecting for edible seed, can utilize these rich datasets, it is necessary to develop common ontologies and accessible ontology platforms. One such platform, Crop Ontology, was created in 2008 by the Consortium of International Agricultural Research Centers (CGIAR) to host crop-specific trait ontologies that support standardized plant breeding databases. In the present study, we describe the sainfoin crop ontology (CO). An in-depth literature review was performed to develop a comprehensive list of traits measured and reported in sainfoin. Because the same traits can be measured in different ways, ultimately, a set of 98 variables (variable = plant trait + method of measurement + scale of measurement) used to describe variation in sainfoin were identified. Variables were formatted and standardized based on guidelines provided here for inclusion in the sainfoin CO. The 98 variables contained a total of 82 traits from four trait classes of which 24 were agronomic, 31 were morphological, 19 were seed and forage quality related, and 8 were phenological. In addition to the developed variables, we have provided a roadmap for developing and submission of new traits to the sainfoin CO.

Re-imagining crop domestication in the era of high throughput phenomics.

De novo domestication is an exciting option for increasing species diversity and ecosystem service functionality of agricultural landscapes. Genomic selection (GS), the application of genomic markers to predict phenotypic traits in a breeding population, offers the possibility of rapid genetic improvement, making GS especially attractive for modifying traits of long-lived species. However, for some wild species just entering the domestication pipeline, especially those with large and complex genomes, a lack of funding and/or prior genome characterization, GS is often out of reach. High throughput phenomics has the potential to augment traditional pedigree selection, reduce costs and amplify impacts of genomic selection, and even create new predictive selection approaches independent of sequencing or pedigrees.

The role of genus and life span in predicting seed and vegetative trait variation and correlation in Lathyrus, Phaseolus, and Vicia.

PREMISE: Annual and perennial life history transitions are abundant among angiosperms, and understanding the phenotypic variation underlying life span shifts is a key endeavor of plant evolutionary biology. Comparative analyses of trait variation and correlation networks among annual and perennial plants is increasingly important as new herbaceous perennial crops are being developed for edible seed. However, it remains unclear how seed to vegetative growth trait relationships correlate with life span. METHODS: To assess the relative roles of genus and life span in predicting phenotypic variation and trait correlations, we measured seed size and shape, germination proportion, and early-life-stage plant height and leaf growth over 3 mo in 29 annual and perennial, herbaceous congeneric species from three legume genera (Lathyrus, Phaseolus, and Vicia). RESULTS: Genus was the strongest predictor of seed size and shape variation, and life span consistently predicted plant height and leaf number at single time points. Correlation networks revealed that annual species had significant associations between seed traits and vegetative traits, whereas perennials had no significant seed-vegetative associations. Each genus also differed in the extent of integration between seed and vegetative traits, as well as within-vegetative-trait correlation patterns. CONCLUSIONS: Genus and life span were important for predicting aspects of early-life-stage phenotypic variation and trait relationships. Differences in phenotypic correlation may indicate that selection on seed size traits will impact vegetative growth differently depending on life span, which has important implications for nascent perennial breeding programs.

Evaluation of pharmacists' awareness of illegal online pharmacies and perceived impact on safe access to medicines.

Background: An estimated 95% of all online pharmacies operate unlawfully. Illegal online pharmacies distribute substandard and falsified medical products that may result in patient harm and suboptimal treatment, leading to an overall mistrust of medications, healthcare providers, and health systems. As medication experts, pharmacists are trusted to guide patients in selection of safe and effective medication therapy. Objective: The objective of this study was to determine gaps in knowledge and recognition of the negative clinical and safety impacts associated with illegal Internet pharmacies by licensed pharmacists. Methods: A 37-question electronic survey was developed and distributed to pharmacists across the United States by email via a database from the American Pharmacists Association. Descriptive statistics was utilized to analyze data. Results: A total of 347 pharmacists from across the United States responded to at least one question in the survey. In all, 58% of pharmacists reported a lack of confidence in their ability to counsel patients on the identification of illegal pharmacy websites. Fewer than 60% of pharmacists were able to accurately identify the legitimacy of a webpage based on visual characteristics. In addition, 75% of pharmacists reported being unfamiliar with resources available to help consumers identify safe and legitimate online pharmacies. Conclusion: Integration of the topic into pharmacy education curricula, training on available resources, and additional research into the prevalence and impact of illegal pharmacy websites are necessary to ensure that pharmacists and other healthcare professionals are adequately prepared to protect their communities from the threat of illegal online pharmacies.

Correction: Integrating transcriptomic network reconstruction and eQTL analyses reveals mechanistic connections between genomic architecture and Brassica rapa development.

[This corrects the article DOI: 10.1371/journal.pgen.1008367.].

A Nested Association Mapping Panel in Arabidopsis thaliana for Mapping and Characterizing Genetic Architecture.

Linkage and association mapping populations are crucial public resources that facilitate the characterization of trait genetic architecture in natural and agricultural systems. We define a large nested association mapping panel (NAM) from 14 publicly available recombinant inbred line populations (RILs) of Arabidopsis thaliana, which share a common recurrent parent (Col-0). Using a genotype-by-sequencing approach (GBS), we identified single nucleotide polymorphisms (SNPs; range 563-1525 per population) and subsequently built updated linkage maps in each of the 14 RIL sets. Simulations in individual RIL populations indicate that our GBS markers have improved power to detect small effect QTL and enhanced resolution of QTL support intervals in comparison to original linkage maps. Using these robust linkage maps, we imputed a common set of publicly available parental SNPs into each RIL linkage map, generating overlapping markers across all populations. Though ultimately depending on allele frequencies at causal loci, simulations of the NAM panel suggest that surveying between 4 to 7 of the 14 RIL populations provides high resolution of the genetic architecture of complex traits, relative to a single mapping population.

New Food Crop Domestication in the Age of Gene Editing: Genetic, Agronomic and Cultural Change Remain Co-evolutionarily Entangled.

The classic domestication scenario for grains and fruits has been portrayed as the lucky fixation of major-effect "domestication genes." Characterization of these genes plus recent improvements in generating novel alleles (e.g., by gene editing) have created great interest in de novo domestication of new crops from wild species. While new gene editing technologies may accelerate some genetic aspects of domestication, we caution that de novo domestication should be understood as an iterative process rather than a singular event. Changes in human social preferences and relationships and ongoing agronomic innovation, along with broad genetic changes, may be foundational. Allele frequency changes at many loci controlling quantitative traits not normally included in the domestication syndrome may be required to achieve sufficient yield, quality, defense, and broad adaptation. The environments, practices and tools developed and maintained by farmers and researchers over generations contribute to crop yield and success, yet those may not be appropriate for new crops without a history of agronomy. New crops must compete with crops that benefit from long-standing participation in human cultural evolution; adoption of new crops may require accelerating the evolution of new crops' culinary and cultural significance, the emergence of markets and trade, and the formation and support of agricultural and scholarly institutions. We provide a practical framework that highlights and integrates these genetic, agronomic, and cultural drivers of change to conceptualize de novo domestication for communities of new crop domesticators, growers and consumers. Major gene-focused domestication may be valuable in creating allele variants that are critical to domestication but will not alone result in widespread and ongoing cultivation of new crops. Gene editing does not bypass or diminish the need for classical breeding, ethnobotanical and horticultural knowledge, local agronomy and crop protection research and extension, farmer participation, and social and cultural research and outreach. To realize the ecological and social benefits that a new era of de novo domestication could offer, we call on funding agencies, proposal reviewers and authors, and research communities to value and support these disciplines and approaches as essential to the success of the breakthroughs that are expected from gene editing techniques.

Comparative Analysis of Early Life Stage Traits in Annual and Perennial Phaseolus Crops and Their Wild Relatives.

Herbaceous perennial species are receiving increased attention for their potential to provide both edible products and ecosystem services in agricultural systems. Many legumes (Fabaceae Lindl.) are of special interest due to nitrogen fixation carried out by bacteria in their roots and their production of protein-rich, edible seeds. However, herbaceous perennial legumes have yet to enter widespread use as pulse crops, and the response of wild, herbaceous perennial species to artificial selection for increased seed yield remains under investigation. Here we compare cultivated and wild accessions of congeneric annual and herbaceous perennial legume species to investigate associations of lifespan and cultivation with early life stage traits including seed size, germination, and first year vegetative growth patterns, and to assess variation and covariation in these traits. We use "cultivated" to describe accessions with a history of human planting and use, which encompasses a continuum of domestication. Analyses focused on three annual and four perennial species of the economically important genus Phaseolus. We found a significant association of both lifespan and cultivation status with seed size (weight, two-dimensional lateral area, length), node number, and most biomass traits (with cultivation alone showing additional significant associations). Wild annual and perennial accessions primarily showed only slight differences in trait values. Relative to wild forms, both cultivated annual and cultivated perennial accessions exhibited greater seed size and larger overall vegetative size, with cultivated perennials showing greater mean trait differences relative to wild accessions than cultivated annuals. Germination proportion was significantly lower in cultivated relative to wild annual accessions, while no significant difference was observed between cultivated and wild perennial germination. Regardless of lifespan and cultivation status, seed size traits were positively correlated with most vegetative traits, and all biomass traits examined here were positively correlated. This study highlights some fundamental similarities and differences between annual and herbaceous perennial legumes and provides insights into how perennial legumes might respond to artificial selection compared to annual species.

Integrating transcriptomic network reconstruction and eQTL analyses reveals mechanistic connections between genomic architecture and Brassica rapa development.

Plant developmental dynamics can be heritable, genetically correlated with fitness and yield, and undergo selection. Therefore, characterizing the mechanistic connections between the genetic architecture governing plant development and the resulting ontogenetic dynamics of plants in field settings is critically important for agricultural production and evolutionary ecology. We use hierarchical Bayesian Function-Valued Trait (FVT) models to estimate Brassica rapa growth curves throughout ontogeny, across two treatments, and in two growing seasons. We find genetic variation for plasticity of growth rates and final sizes, but not the inflection point (transition from accelerating to decelerating growth) of growth curves. There are trade-offs between growth rate and duration, indicating that selection for maximum yields at early harvest dates may come at the expense of late harvest yields and vice versa. We generate eigengene modules and determine which are co-expressed with FVT traits using a Weighted Gene Co-expression Analysis. Independently, we seed a Mutual Rank co-expression network model with FVT traits to identify specific genes and gene networks related to FVT. GO-analyses of eigengene modules indicate roles for actin/cytoskeletal genes, herbivore resistance/wounding responses, and cell division, while MR networks demonstrate a close association between metabolic regulation and plant growth. We determine that combining FVT Quantitative Trait Loci (QTL) and MR genes/WGCNA eigengene expression profiles better characterizes phenotypic variation than any single data type (i.e. QTL, gene, or eigengene alone). Our network analysis allows us to employ a targeted eQTL analysis, which we use to identify regulatory hotspots for FVT. We examine cis vs. trans eQTL that mechanistically link FVT QTL with structural trait variation. Colocalization of FVT, gene, and eigengene eQTL provide strong evidence for candidate genes influencing plant height. The study is the first to explore eQTL for FVT, and specifically do so in agroecologically relevant field settings.

Environmental heterogeneity generates intrapopulation variation in life-history traits in an annual plant.

Environmental variation affects a plant's life cycle by influencing the timing of germination and flowering, and the duration of the growing season. Yet we know little information about how environmental heterogeneity generates variation in germination schedules and the consequences for growth and fecundity through genetic and plastic responses. We use an annual population of Mimulus guttatus in which, in nature, seeds germinate in both fall and spring. We investigate whether there is a genetic basis to the timing of germination, the effect of germination timing on fecundity, and if growth and flowering respond plastically to compensate for different season lengths. Using sibling families grown in simulated seasonal conditions, we find that families do not differ in their propensity to germinate between seasons. However, the germination season affects subsequent growth and flowering time, with significant genotype-by-environment interactions (G × E). Most G × E is due to unequal variance between seasons, because the spring cohort harbours little genetic variance. Despite their different season lengths, the cohorts do not differ in flower number (fecundity). Heterogeneous environments with unpredictable risks may maintain promiscuous germination, which then affects flowering time. Therefore, if selection at particular life stages changes with climate change, there may be consequences for the entire life cycle.

QTL Underlying Circadian Clock Parameters Under Seasonally Variable Field Settings in Arabidopsis thaliana.

The circadian clock facilitates coordination of the internal rhythms of an organism to daily environmental conditions, such as the light-dark cycle of one day. Circadian period length (the duration of one endogenous cycle) and phase (the timing of peak activity) exhibit quantitative variation in natural populations. Here, we measured circadian period and phase in June, July and September in three Arabidopsis thaliana recombinant inbred line populations. Circadian period and phase were estimated from bioluminescence of a genetic construct between a native circadian clock gene (COLD CIRCADIAN RHYTHM RNA BINDING 2) and the reporter gene (LUCIFERASE) after lines were entrained under field settings. Using a Bayesian mapping approach, we estimated the median number and effect size of genomic regions (Quantitative Trait Loci, QTL) underlying circadian parameters and the degree to which these regions overlap across months of the growing season. We also tested for QTL associations between the circadian clock and plant morphology. The genetic architecture of circadian phase was largely independent across months, as evidenced by the fact that QTL determining phase values in one month of the growing season were different from those determining phase in a second month. QTL for circadian parameters were shared with both cauline and rosette branching in at least one mapping population. The results provide insights into the QTL architecture of the clock under field settings, and suggest that the circadian clock is highly responsive to changing environments and that selection can act on clock phase in a nuanced manner.

Assortative mating by flowering time and its effect on correlated traits in variable environments.

Reproductive timing is a key life-history trait that impacts the pool of available mates, the environment experienced during flowering, and the expression of other traits through genetic covariation. Selection on phenology, and its consequences on other life-history traits, has considerable implications in the context of ongoing climate change and shifting growing seasons. To test this, we grew field-collected seed from the wildflower Mimulus guttatus in a greenhouse to assess the standing genetic variation for flowering time and covariation with other traits. We then created full-sib families through phenological assortative mating and grew offspring in three photoperiod treatments representing seasonal variation in daylength. We find substantial quantitative genetic variation for the onset of flowering time, which covaried with vegetative traits. The assortatively-mated offspring varied in their critical photoperiod by over two hours, so that families differed in their probability of flowering across treatments Allocation to flowering and vegetative growth changed across the daylength treatments, with consistent direction and magnitude of covariation among flowering time and other traits. Our results suggest that future studies of flowering time evolution should consider the joint evolution of correlated traits and shifting seasonal selection to understand how environmental variation influences life histories.

The role of cold cues at different life stages on germination and flowering phenology.

PREMISE OF THE STUDY: The timing of major phenological transitions is critical to lifetime fitness, and life history theory predicts differences for annual and perennial plants. To correctly time these transitions, many plants rely on environmental cues such as exposure to extended periods of cold, which may occur at different stages throughout their lifetime. METHODS: We studied the role of cold at different life stages, by jointly exposing seed (stratification) and rosettes (vernalization) to cold. We used 23 populations of Mimulus guttatus, which vary from annuals to perennials, and investigated how cold at one or both stages affected germination, flowering, growth, and biomass. KEY RESULTS: We found that stratification and vernalization interact to affect life cycle transitions, and that cold at either stage could synchronize flowering phenology. For perennials, either stratification or vernalization is necessary for maximum flowering. We also found that germination timing covaried with later traits. Moreover, plants from environments with dissimilar climates displayed different phenological responses to stratification or vernalization. CONCLUSIONS: In general, cold is more important for seed germination in annuals and plants from environments with warm temperatures and variable precipitation. In contrast, cold is more important for flowering in perennials: it accelerates flowering in plants from lower precipitation environments, and it increases flowering proportion in plants from cooler, more stable precipitation environments. We discuss our findings in the context of the variable environments plants experience within a population and the variation encountered across the biogeographic native range of the species.

Circadian rhythms are associated with shoot architecture in natural settings.

Circadian rhythms are key regulators of diverse biological processes under controlled settings. Yet, the phenotypic and fitness consequences of quantitative variation in circadian rhythms remain largely unexplored in the field. As with other pathways, phenotypic characterization of circadian outputs in the field may reveal novel clock functions. Across consecutive growing seasons, we test for associations between clock variation and flowering phenology, plant size, shoot architecture, and fruit set in clock mutants and segregating progenies of Arabidopsis thaliana expressing quantitative variation in circadian rhythms. Using structural equation modeling, we find that genotypic variation in circadian rhythms within a growing season is associated directly with branching, which in turn affects fruit production. Consistent with direct associations between the clock and branching in segregating progenies, cauline branch number is lower and rosette branch number higher in a short-period mutant relative to wild-type and long-period genotypes, independent of flowering time. Differences in branching arise from variation in meristem fate as well as leaf production rate before flowering and attendant increases in meristem number. Our results suggest that clock variation directly affects shoot architecture in the field, suggesting a novel clock function and means by which the clock affects performance.

Circadian rhythms are associated with variation in photosystem II function and photoprotective mechanisms.

The circadian clock regulates many aspects of leaf gas supply and biochemical demand for CO2 , and is hypothesized to improve plant performance. Yet the extent to which the clock may regulate the efficiency of photosystem II (PSII) and photoprotective mechanisms such as heat dissipation is less explored. Based on measurements of chlorophyll a fluorescence, we estimated the maximum efficiency of PSII in light (Fv'/Fm') and heat dissipation by nonphotochemical quenching (NPQ). We further dissected total NPQ into its main components, qE (pH-dependent quenching), qT (state-transition quenching), and qI (quenching related to photoinhibition), in clock mutant genotypes of Arabidopsis thaliana, the cognate wild-type genotypes, and a panel of recombinant inbred lines expressing quantitative variation in clock period. Compared with mutants with altered clock function, we observed that wild-type genotypes with clock period lengths of approximately 24 hr had both higher levels of Fv'/Fm', indicative of improved PSII function, and reduced NPQ, suggestive of lower stress on PSII light harvesting complexes. In the recombinant inbred lines, genetic variances were significant for Fv'/Fm' and all 3 components of NPQ, with qE explaining the greatest proportion of NPQ. Bivariate tests of association and structural equation models of hierarchical trait relationships showed that quantitative clock variation was empirically associated with Fv'/Fm' and NPQ, with qE mediating the relationship with gas exchange. The results demonstrate significant segregating variation for all photoprotective components, and suggest the adaptive significance of the clock may partly derive from its regulation of the light reactions of photosynthesis and of photoprotective mechanisms.

Bayesian estimation and use of high-throughput remote sensing indices for quantitative genetic analyses of leaf growth.

KEY MESSAGE: We develop Bayesian function-valued trait models that mathematically isolate genetic mechanisms underlying leaf growth trajectories by factoring out genotype-specific differences in photosynthesis. Remote sensing data can be used instead of leaf-level physiological measurements. Characterizing the genetic basis of traits that vary during ontogeny and affect plant performance is a major goal in evolutionary biology and agronomy. Describing genetic programs that specifically regulate morphological traits can be complicated by genotypic differences in physiological traits. We describe the growth trajectories of leaves using novel Bayesian function-valued trait (FVT) modeling approaches in Brassica rapa recombinant inbred lines raised in heterogeneous field settings. While frequentist approaches estimate parameter values by treating each experimental replicate discretely, Bayesian models can utilize information in the global dataset, potentially leading to more robust trait estimation. We illustrate this principle by estimating growth asymptotes in the face of missing data and comparing heritabilities of growth trajectory parameters estimated by Bayesian and frequentist approaches. Using pseudo-Bayes factors, we compare the performance of an initial Bayesian logistic growth model and a model that incorporates carbon assimilation (A max) as a cofactor, thus statistically accounting for genotypic differences in carbon resources. We further evaluate two remotely sensed spectroradiometric indices, photochemical reflectance (pri2) and MERIS Terrestrial Chlorophyll Index (mtci) as covariates in lieu of A max, because these two indices were genetically correlated with A max across years and treatments yet allow much higher throughput compared to direct leaf-level gas-exchange measurements. For leaf lengths in uncrowded settings, including A max improves model fit over the initial model. The mtci and pri2 indices also outperform direct A max measurements. Of particular importance for evolutionary biologists and plant breeders, hierarchical Bayesian models estimating FVT parameters improve heritabilities compared to frequentist approaches.

Circadian rhythms vary over the growing season and correlate with fitness components.

Circadian clocks have evolved independently in all three domains of life, suggesting that internal mechanisms of time-keeping are adaptive in contemporary populations. However, the performance consequences of either discrete or quantitative clock variation have rarely been tested in field settings. Clock sensitivity of diverse segregating lines to the environment remains uncharacterized as do the statistical genetic parameters that determine evolutionary potential. In field studies with Arabidopsis thaliana, we found that major perturbations to circadian cycle length (referred to as clock period) via mutation reduce both survival and fecundity. Subtler adjustments via genomic introgression of naturally occurring alleles indicated that clock periods slightly >24 hr were adaptive, consistent with prior models describing how well the timing of biological processes is adjusted within a diurnal cycle (referred to as phase). In segregating recombinant inbred lines (RILs), circadian phase varied up to 2 hr across months of the growing season, and both period and phase expressed significant genetic variances. Performance metrics including developmental rate, size and fruit set were described by principal components (PC) analyses and circadian parameters correlated with the first PC, such that period lengths slightly >24 hr were associated with improved performance in multiple RIL sets. These experiments translate functional analyses of clock behaviour performed in controlled settings to natural ones, demonstrating that quantitative variation in circadian phase is highly responsive to seasonally variable abiotic factors. The results expand upon prior studies in controlled settings, showing that discrete and quantitative variation in clock phenotypes correlates with performance in nature.

Epistasis × environment interactions among Arabidopsis thaliana glucosinolate genes impact complex traits and fitness in the field.

Despite the growing number of studies showing that genotype × environment and epistatic interactions control fitness, the influences of epistasis × environment interactions on adaptive trait evolution remain largely uncharacterized. Across three field trials, we quantified aliphatic glucosinolate (GSL) defense chemistry, leaf damage, and relative fitness using mutant lines of Arabidopsis thaliana varying at pairs of causal aliphatic GSL defense genes to test the impact of epistatic and epistasis × environment interactions on adaptive trait variation. We found that aliphatic GSL accumulation was primarily influenced by additive and epistatic genetic variation, leaf damage was primarily influenced by environmental variation and relative fitness was primarily influenced by epistasis and epistasis × environment interactions. Epistasis × environment interactions accounted for up to 48% of the relative fitness variation in the field. At a single field site, the impact of epistasis on relative fitness varied significantly over 2 yr, showing that epistasis × environment interactions within a location can be temporally dynamic. These results suggest that the environmental dependency of epistasis can profoundly influence the response to selection, shaping the adaptive trajectories of natural populations in complex ways, and deserves further consideration in future evolutionary studies.

Genetic architecture, biochemical underpinnings and ecological impact of floral UV patterning.

Floral attraction traits can significantly affect pollinator visitation patterns, but adaptive evolution of these traits may be constrained by correlations with other traits. In some cases, molecular pathways contributing to floral attraction are well characterized, offering the opportunity to explore loci potentially underlying variation among individuals. Here, we quantify the range of variation in floral UV patterning (i.e. UV 'bulls-eye nectar guides) among crop and wild accessions of Brassica rapa. We then use experimental crosses to examine the genetic architecture, candidate loci and biochemical underpinnings of this patterning as well as phenotypic manipulations to test the ecological impact. We find qualitative variation in UV patterning between wild (commonly lacking UV patterns) and crop (commonly exhibiting UV patterns) accessions. Similar to the majority of crops, recombinant inbred lines (RILs) derived from an oilseed crop × WI fast-plant® cross exhibit UV patterns, the size of which varies extensively among genotypes. In RILs, we further observe strong statistical-genetic and QTL correlations within petal morphological traits and within measurements of petal UV patterning; however, correlations between morphology and UV patterning are weak or nonsignificant, suggesting that UV patterning is regulated and may evolve independently of overall petal size. HPLC analyses reveal a high concentration of sinapoyl glucose in UV-absorbing petal regions, which, in concert with physical locations of UV-trait QTLs, suggest a regulatory and structural gene as candidates underlying observed quantitative variation. Finally, insects prefer flowers with UV bulls-eye patterns over those that lack patterns, validating the importance of UV patterning in pollen-limited populations of B. rapa.