Genotype and phenotype data standardization, utilization and integration in the big data era for agricultural sciences.

Large-scale genotype and phenotype data have been increasingly generated to identify genetic markers, understand gene function and evolution and facilitate genomic selection. These datasets hold immense value for both current and future studies, as they are vital for crop breeding, yield improvement and overall agricultural sustainability. However, integrating these datasets from heterogeneous sources presents significant challenges and hinders their effective utilization. We established the Genotype-Phenotype Working Group in November 2021 as a part of the AgBioData Consortium (https://www.agbiodata.org) to review current data types and resources that support archiving, analysis and visualization of genotype and phenotype data to understand the needs and challenges of the plant genomic research community. For 2021-22, we identified different types of datasets and examined metadata annotations related to experimental design/methods/sample collection, etc. Furthermore, we thoroughly reviewed publicly funded repositories for raw and processed data as well as secondary databases and knowledgebases that enable the integration of heterogeneous data in the context of the genome browser, pathway networks and tissue-specific gene expression. Based on our survey, we recommend a need for (i) additional infrastructural support for archiving many new data types, (ii) development of community standards for data annotation and formatting, (iii) resources for biocuration and (iv) analysis and visualization tools to connect genotype data with phenotype data to enhance knowledge synthesis and to foster translational research. Although this paper only covers the data and resources relevant to the plant research community, we expect that similar issues and needs are shared by researchers working on animals. Database URL: https://www.agbiodata.org.

SPOT: Scanning plant IoT facility for high-throughput plant phenotyping.

Many plant phenotyping platforms have been kept out of the reach of smaller labs and institutions due to high cost and proprietary software. The Scanning Plant IoT (SPOT) Facility, located at the University of Florida, is a mobile, laboratory-based platform that facilitates open-source collection of high-quality, interoperable plant phenotypic data. It consists of three main sensors: a hyperspectral sensor, a thermal camera, and a LiDAR camera. Real-time data from the sensors can be collected in its 10 ft. × 10 ft. scanning region. The mobility of the device allows its use in large growth chambers, environmentally controlled rooms, or greenhouses. Sensors are oriented nadir and positioned via computer numerical control of stepper motors. In a preliminary experiment, data gathered from SPOT was used to autonomously and nondestructively differentiate between cultivars.

Dynamic QTL-based ecophysiological models to predict phenotype from genotype and environment data.

BACKGROUND: Predicting the phenotype from the genotype is one of the major contemporary challenges in biology. This challenge is greater in plants because their development occurs mostly post-embryonically under diurnal and seasonal environmental fluctuations. Most current crop simulation models are physiology-based models capable of capturing environmental fluctuations but cannot adequately capture genotypic effects because they were not constructed within a genetics framework. RESULTS: We describe the construction of a mixed-effects dynamic model to predict time-to-flowering in the common bean (Phaseolus vulgaris L.). This prediction model applies the developmental approach used by traditional crop simulation models, uses direct observational data, and captures the Genotype, Environment, and Genotype-by-Environment effects to predict progress towards time-to-flowering in real time. Comparisons to a traditional crop simulation model and to a previously developed static model shows the advantages of the new dynamic model. CONCLUSIONS: The dynamic model can be applied to other species and to different plant processes. These types of models can, in modular form, gradually replace plant processes in existing crop models as has been implemented in BeanGro, a crop simulation model within the DSSAT Cropping Systems Model. Gene-based dynamic models can accelerate precision breeding of diverse crop species, particularly with the prospects of climate change. Finally, a gene-based simulation model can assist policy decision makers in matters pertaining to prediction of food supplies.

Glycerol-Based Dendrimer Nanocomposite Film as a Tunable pH-Sensor for Food Packaging.

Large amounts of food are wasted during the food supply chain. This loss is in part due to consumer confusion over dates on food packages that can indicate a variety of quality indicators in the product (e.g., expiration date, "best by" date, "sell by" dates, etc.). To reduce this food loss, much research has been focused on the films that offer simple and easily manipulated indication systems to detect food spoilage. However, these materials are usually hydrophilic biopolymers that can detect the food spoilage in a wide pH range but do not provide highly sensitive real-time measurements. In this work, a glycerol-based nanocomposite core-shell latex film was synthesized to create a responsive packaging material that can provide real-time pH detection of food with high sensitivity. First, the pH-responsive dendrimer comonomer was synthesized from glycerol and diamine. Then, the nanoencapsulation polymerization process via miniemulsion was conducted to form a core-shell structure with tunable nanoshell thickness for a sensible pH-responsive release (<0.5 pH change). Next, the flexible film encapsulated a color-indicative dye that provided highly sensitive and visible color changes as both the pH dropped and the time elapsed in the food. This film also provided a barrier to water and heat and resisted deformation. Ultimately, this nanocomposite flexible film pending a pH sensor has the potential as an intelligent food packaging material for a universal, accurate, easy-to-use, and real-time food spoilage monitoring system to reduce food waste.

A Predictive Model for Time-to-Flowering in the Common Bean Based on QTL and Environmental Variables.

The common bean is a tropical facultative short-day legume that is now grown in tropical and temperate zones. This observation underscores how domestication and modern breeding can change the adaptive phenology of a species. A key adaptive trait is the optimal timing of the transition from the vegetative to the reproductive stage. This trait is responsive to genetically controlled signal transduction pathways and local climatic cues. A comprehensive characterization of this trait can be started by assessing the quantitative contribution of the genetic and environmental factors, and their interactions. This study aimed to locate significant QTL (G) and environmental (E) factors controlling time-to-flower in the common bean, and to identify and measure G × E interactions. Phenotypic data were collected from a biparental [Andean × Mesoamerican] recombinant inbred population (F11:14, 188 genotypes) grown at five environmentally distinct sites. QTL analysis using a dense linkage map revealed 12 QTL, five of which showed significant interactions with the environment. Dissection of G × E interactions using a linear mixed-effect model revealed that temperature, solar radiation, and photoperiod play major roles in controlling common bean flowering time directly, and indirectly by modifying the effect of certain QTL. The model predicts flowering time across five sites with an adjusted r-square of 0.89 and root-mean square error of 2.52 d. The model provides the means to disentangle the environmental dependencies of complex traits, and presents an opportunity to identify in silico QTL allele combinations that could yield desired phenotypes under different climatic conditions.

Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight.

PREMISE OF THE STUDY: Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. METHODS: We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). KEY RESULTS: In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. CONCLUSIONS: A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants.

Development of a QTL-environment-based predictive model for node addition rate in common bean.

KEY MESSAGE: This work reports the effects of the genetic makeup, the environment and the genotype by environment interactions for node addition rate in an RIL population of common bean. This information was used to build a predictive model for node addition rate. To select a plant genotype that will thrive in targeted environments it is critical to understand the genotype by environment interaction (GEI). In this study, multi-environment QTL analysis was used to characterize node addition rate (NAR, node day- 1) on the main stem of the common bean (Phaseolus vulgaris L). This analysis was carried out with field data of 171 recombinant inbred lines that were grown at five sites (Florida, Puerto Rico, 2 sites in Colombia, and North Dakota). Four QTLs (Nar1, Nar2, Nar3 and Nar4) were identified, one of which had significant QTL by environment interactions (QEI), that is, Nar2 with temperature. Temperature was identified as the main environmental factor affecting NAR while day length and solar radiation played a minor role. Integration of sites as covariates into a QTL mixed site-effect model, and further replacing the site component with explanatory environmental covariates (i.e., temperature, day length and solar radiation) yielded a model that explained 73% of the phenotypic variation for NAR with root mean square error of 16.25% of the mean. The QTL consistency and stability was examined through a tenfold cross validation with different sets of genotypes and these four QTLs were always detected with 50-90% probability. The final model was evaluated using leave-one-site-out method to assess the influence of site on node addition rate. These analyses provided a quantitative measure of the effects on NAR of common beans exerted by the genetic makeup, the environment and their interactions.

Transcriptome analyses of Arabidopsis thaliana seedlings grown in space: implications for gravity-responsive genes.

The transcriptome of seedlings was analyzed from experiments performed on the International Space Station to study the interacting effects of light and gravity on plant tropisms (project named TROPI-2; Kiss et al. 2012). Seeds of Arabidopsis were germinated in space, and seedlings were then grown in the European Modular Cultivation System for 4 days at ~1g followed by exposure to a range of gravitational accelerations (from microgravity to 1g) and two light treatments (blue light with or without a 1 h pretreatment with red). At the end of the experiments, the cassettes containing the seedlings were frozen in the minus eighty laboratory freezer and returned to Earth on space shuttle mission STS-131. The RNA was extracted from whole seedlings and used for the transcriptome analyses. A comparison of 1g spaceflight samples with 1g ground controls identified 230 genes that were differentially regulated at least twofold, emphasizing the need for "in situ" tissue fixation on a 1g centrifuge as an important control for spaceflight experiments. A further comparison of all spaceflight samples with ground controls identified approximately 280 genes that were differentially regulated at least twofold. Of these genes, several were involved in regulating cell polarity (i.e., auxin, calcium, lipid metabolism), cell-wall development, oxygen status, and cell defense or stress. However, when the transcriptome of the all g-treated spaceflight samples was compared with microgravity samples, only ~130 genes were identified as being differently regulated (P ≤ 0.01). Of this subset, only 27 genes were at least twofold differently regulated between microgravity and 1g space samples and included putative/pseudo/undefined genes (14), transposable elements (5), an expansin (ATEXP24; At1g21240), a cell-wall kinase (WAK3; At1g21240), a laccase-like flavonoid oxidase (TT10; At5g48100), among others.

In vitro gastric and intestinal digestions of pulsed light-treated shrimp extracts.

Pulsed ultraviolet light (PUV), a novel technology most commonly used for microbial inactivation, has recently been employed to effectively mitigate food allergens in peanuts, soybean, shrimp, and almond. Putative mechanisms for the efficacy of PUV in reducing allergen reactivity include photothermal, photochemical, and photophysical effects. To date, there are no published data highlighting the effects of in vitro simulated gastric and intestinal digestion on the stability of PUV reduced allergen reactivity of food. In this study, PUV-treated shrimp extracts were subjected to simulated gastric fluid containing pepsin and simulated intestinal fluid containing trypsin and chymotrypsin, and then tested for changes in allergen potency. SDS-PAGE showed no major band deviation between undigested and digested PUV-treated shrimp extracts. IgE binding to tropomyosin remained markedly decreased as seen in Western blot analysis. Total shrimp allergen reactivity remained unchanged following in vitro peptic digestion and was markedly reduced following in vitro intestinal digestion as illustrated in indirect ELISA. The PUV reduced shrimp allergens remained at a low level under the in vitro simulated digestive conditions. The results inferred that PUV could be a potential method to create less allergenic shrimp products that would remain at a low allergen level under human gastric and intestinal digestive conditions.

Bioluminescent enzyme assay for the indication of plant stress in enclosed life support systems.

The application of bioluminescent sensors for monitoring key metabolites and enzymes that are indicators of stress in plants is demonstrated. The sensitivity of bioluminescent assay for NAD(P)H and NAD(P)(+) was about 0.5 and 1 nmol, respectively. The levels of NAD(P)H and NAD(P)(+) in radish (Raphanus sativus) root extracts from controls and from stress-induced conditions were compared. To induce environmental stress, the plants were grown in enclosed environmental chambers with low pressure (9 or 32 kPa), high humidity (>80%) and low oxygen partial pressure (down to 3.3-6.5 kPa). The concentrations of NAD(P)(+) and NAD(P)H in plants varied under stress conditions. Decreasing both total pressure from 101.5 to 32 or 9 kPa and partial pressure of oxygen increased the ratio of NAD(P)(+) /NAD(P)H from 0.2 to 4 or 6, respectively. The increase in this ratio suggests that plants are undergoing stress in these hypobaric environments. The developed bioluminescent assay for quantification of pyridine nucleotides in plant tissues is rapid, low-cost and easily performed.

A novel phototropic response to red light is revealed in microgravity.

The aim of this study was to investigate phototropism in plants grown in microgravity conditions without the complications of a 1-g environment. Experiments performed on the International Space Station (ISS) were used to explore the mechanisms of both blue-light- and red-light-induced phototropism in plants. This project utilized the European Modular Cultivation System (EMCS), which has environmental controls for plant growth as well as centrifuges for gravity treatments used as a 1-g control. Images captured from video tapes were used to analyze the growth, development, and curvature of Arabidopsis thaliana plants that developed from seed in space. A novel positive phototropic response to red light was observed in hypocotyls of seedlings that developed in microgravity. This response was not apparent in seedlings grown on Earth or in the 1-g control during the space flight. In addition, blue-light-based phototropism had a greater response in microgravity compared with the 1-g control. Although flowering plants are generally thought to lack red light phototropism, our data suggest that at least some flowering plants may have retained a red light sensory system for phototropism. Thus, this discovery may have important implications for understanding the evolution of light sensory systems in plants.

Extraction and labeling methods for microarrays using small amounts of plant tissue.

Procedures were developed to maximize the yield of high-quality RNA from small amounts of plant biomass for microarrays. Two disruption techniques (bead milling and pestle and mortar) were compared for the yield and the quality of RNA extracted from 1-week-old Arabidopsis thaliana seedlings (approximately 0.5-30 mg total biomass). The pestle and mortar method of extraction showed enhanced RNA quality at the smaller biomass samples compared with the bead milling technique, although the quality in the bead milling could be improved with additional cooling steps. The RNA extracted from the pestle and mortar technique was further tested to determine if the small quantity of RNA (500 ng-7 microg) was appropriate for microarray analyses. A new method of low-quantity RNA labeling for microarrays (NuGEN Technologies, Inc.) was used on five 7-day-old seedlings (approximately 2.5 mg fresh weight total) of Arabidopsis that were grown in the dark and exposed to 1 h of red light or continued dark. Microarray analyses were performed on a small plant sample (five seedlings; approximately 2.5 mg) using these methods and compared with extractions performed with larger biomass samples (approximately 500 roots). Many well-known light-regulated genes between the small plant samples and the larger biomass samples overlapped in expression changes, and the relative expression levels of selected genes were confirmed with quantitative real-time polymerase chain reaction, suggesting that these methods can be used for plant experiments where the biomass is extremely limited (i.e. spaceflight studies).

Gene profiling of the red light signalling pathways in roots.

Red light, acting through the phytochromes, controls numerous aspects of plant development. Many of the signal transduction elements downstream of the phytochromes have been identified in the aerial portions of the plant; however, very few elements in red-light signalling have been identified specifically for roots. Gene profiling studies using microarrays and quantitative Real-Time PCR were performed to characterize gene expression changes in roots of Arabidopsis seedlings exposed to 1 h of red light. Several factors acting downstream of phytochromes in red-light signalling in roots were identified. Some of the genes found to be differentially expressed in this study have already been characterized in the red-light-signalling pathway for whole plants. For example, PHYTOCHROME KINASE 1 (PKS1), LONG HYPOCOTYL 5 (HY5), EARLY FLOWERING 4 (ELF4), and GIGANTEA (GI) were all significantly up-regulated in roots of seedlings exposed to 1 h of red light. The up-regulation of SUPPRESSOR OF PHYTOCHROME A RESPONSES 1 (SPA1) and CONSTITUTIVE PHOTOMORPHOGENIC 1-like (COP1-like) genes suggests that the PHYA-mediated pathway was attenuated by red light. In addition, genes involved in lateral root and root hair formation, root plastid development, phenylpropanoid metabolism, and hormone signalling were also regulated by exposure to red light. Interestingly, members of the RPT2/NPH3 (ROOT PHOTOTROPIC 2/NON PHOTOTROPIC HYPOCOTYL 3) family, which have been shown to mediate blue-light-induced phototropism, were also differentially regulated in roots in red light. Therefore, these results suggest that red and blue light pathways interact in roots of seedlings and that many elements involved in red-light-signalling found in the aerial portions of the plant are differentially expressed in roots within 1 h of red light exposure.

The roles of phytochromes in elongation and gravitropism of roots.

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.

Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots.

Phototropism as well as gravitropism plays a role in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism, but red light induces positive phototropism in Arabidopsis roots. Phytochrome A (phyA) and phyB mediate the positive red-light-based photoresponse in roots since single mutants (and the double phyAB mutant) were severely impaired in this response. In blue-light-based negative phototropism, phyA and phyAB (but not phyB) were inhibited in the response relative to the WT. In root gravitropism, phyB and phyAB (but not phyA) were inhibited in the response compared to the WT. The differences observed in tropistic responses were not due to growth limitations since the growth rates among all the mutants tested were not significantly different from that of the WT. Thus, our study shows that the blue-light and red-light systems interact in roots and that phytochrome plays a key role in plant development by integrating multiple environmental stimuli.

Root phototropism: how light and gravity interact in shaping plant form.

The interactions among tropisms can be critical in determining the final growth form of plants and plant organs. We have studied tropistic responses in roots as an example of these type of interactions. While gravitropism is the predominant tropistic response in roots, phototropism also plays a role in the oriented growth in this organ in flowering plants. In blue or white light, roots exhibit negative phototropism, but red light induces positive phototropism. In the flowering plant Arabidopsis, the photosensitive pigments phytochrome A (phyA) and phytochrome B (phyB) mediate this positive red-light-based photoresponse in roots since single mutants (and the double phyAB mutant) were severely impaired in this response. While blue-light-based negative phototropism is primarily mediated by the phototropin family of photoreceptors, the phyA and phyAB mutants (but not phyB) were inhibited in this response relative to the WT. The differences observed in phototropic responses were not due to growth limitations since the growth rates among all the mutants tested were not significantly different from that of the WT. Thus, our study shows that the blue-light and red-light systems interact in plants and that phytochrome plays a key role in integrating multiple environmental stimuli.

Phytochromes A and B mediate red-light-induced positive phototropism in roots.

The interaction of tropisms is important in determining the final growth form of the plant body. In roots, gravitropism is the predominant tropistic response, but phototropism also plays a role in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism that is mediated by the phototropin family of photoreceptors. In contrast, red light induces a positive phototropism in Arabidopsis roots. Because this red-light-induced response is weak relative to both gravitropism and negative phototropism, we used a novel device to study phototropism without the complications of a counteracting gravitational stimulus. This device is based on a computer-controlled system using real-time image analysis of root growth and a feedback-regulated rotatable stage. Our data show that this system is useful to study root phototropism in response to red light, because in wild-type roots, the maximal curvature detected with this apparatus is 30 degrees to 40 degrees, compared with 5 degrees to 10 degrees without the feedback system. In positive root phototropism, sensing of red light occurs in the root itself and is not dependent on shoot-derived signals resulting from light perception. Phytochrome (Phy)A and phyB were severely impaired in red-light-induced phototropism, whereas the phyD and phyE mutants were normal in this response. Thus, PHYA and PHYB play a key role in mediating red-light-dependent positive phototropism in roots. Although phytochrome has been shown to mediate phototropism in some lower plant groups, this is one of the few reports indicating a phytochrome-dependent phototropism in flowering plants.

Interactions between gravitropism and phototropism in plants.

To receive adequate light and nutrients for survival, plants orient stems and stem-like organs toward light and away from the gravity vector and, conversely, orient roots into the soil, away from light toward the direction of gravity. Therefore, both gravity and light can influence the differential growth of plant organs. To add to the complexity of the interactions between gravity and light, each stimulus can enhance or reduce the effectiveness of the other. On earth, the constant presence of gravity makes it difficult to determine whether plant growth and development is influenced by gravity or light alone or the combination of the two stimuli. In the past decade, our understanding of the gravity and light transduction pathways has advanced through the use of mutants in either gravitropic or phototropic responses and the use of innovative techniques that reduce the effects of one stimulus on the other. Thus, both unique and common elements in the transduction pathways of the gravitropic and phototropic responses have been isolated. This article is focused on the interactions between the light- and gravity-transduction pathways and describes methods used to separate the influences of these two environmental stimuli.