Root Pulling Force Across Drought in Maize Reveals Genotype by Environment Interactions and Candidate Genes.

High-throughput, field-based characterization of root systems for hundreds of genotypes in thousands of plots is necessary for breeding and identifying loci underlying variation in root traits and their plasticity. We designed a large-scale sampling of root pulling force, the vertical force required to extract the root system from the soil, in a maize diversity panel under differing irrigation levels for two growing seasons. We then characterized the root system architecture of the extracted root crowns. We found consistent patterns of phenotypic plasticity for root pulling force for a subset of genotypes under differential irrigation, suggesting that root plasticity is predictable. Using genome-wide association analysis, we identified 54 SNPs as statistically significant for six independent root pulling force measurements across two irrigation levels and four developmental timepoints. For every significant GWAS SNP for any trait in any treatment and timepoint we conducted post hoc tests for genotype-by-environment interaction, using a mixed model ANOVA. We found that 8 of the 54 SNPs showed significant GxE. Candidate genes underlying variation in root pulling force included those involved in nutrient transport. Although they are often treated separately, variation in the ability of plant roots to sense and respond to variation in environmental resources including water and nutrients may be linked by the genes and pathways underlying this variation. While functional validation of the identified genes is needed, our results expand the current knowledge of root phenotypic plasticity at the whole plant and gene levels, and further elucidate the complex genetic architecture of maize root systems.

Quantitative trait loci controlling agronomic and biochemical traits in Cannabis sativa.

Understanding the genetic basis of complex traits is a fundamental goal of evolutionary genetics. Yet, the genetics controlling complex traits in many important species such as hemp (Cannabis sativa) remain poorly investigated. Because hemp's change in legal status with the 2014 and 2018 U.S. Federal Farm Bills, interest in the genetics controlling its numerous agriculturally important traits has steadily increased. To better understand the genetics of agriculturally important traits in hemp, we developed an F2 population by crossing two phenotypically distinct hemp cultivars (Carmagnola and USO31). Using whole-genome sequencing, we mapped quantitative trait loci (QTL) associated with variation in numerous agronomic and biochemical traits. A total of 69 loci associated with agronomic (34) and biochemical (35) trait variation were identified. We found that most QTL co-localized, suggesting that the phenotypic distinctions between Carmagnola and USO31 are largely controlled by a small number of loci. We identified TINY and olivetol synthase as candidate genes underlying co-localized QTL clusters for agronomic and biochemical traits, respectively. We functionally validated the olivetol synthase candidate by expressing the alleles in yeast. Gas chromatography-mass spectrometry assays of extracts from these yeast colonies suggest that the USO31 olivetol synthase is functionally less active and potentially explains why USO31 produces lower cannabinoids compared to Carmagnola. Overall, our results help modernize the genomic understanding of complex traits in hemp.

Deployment of Lidar from a Ground Platform: Customizing a Low-Cost, Information-Rich and User-Friendly Application for Field Phenomics Research.

Using sensors and electronic systems for characterization of plant traits provides valuable digital inputs to support complex analytical modeling in genetics research. In field applications, frequent sensor deployment enables the study of the dynamics of these traits and their interaction with the environment. This study focused on implementing lidar (light detection and ranging) technology to generate 2D displacement data at high spatial resolution and extract plant architectural parameters, namely canopy height and cover, in a diverse population of 252 maize (Zea mays L.) genotypes. A prime objective was to develop the mechanical and electrical subcomponents for field deployment from a ground vehicle. Data reduction approaches were implemented for efficient same-day post-processing to generate by-plot statistics. The lidar system was successfully deployed six times in a span of 42 days. Lidar data accuracy was validated through independent measurements in a subset of 75 experimental units. Manual and lidar-derived canopy height measurements were compared resulting in root mean square error (RMSE) = 0.068 m and r2 = 0.81. Subsequent genome-wide association study (GWAS) analyses for quantitative trait locus (QTL) identification and comparisons of genetic correlations and heritabilities for manual and lidar-based traits showed statistically significant associations. Low-cost, field-ready lidar of computational simplicity make possible timely phenotyping of diverse populations in multiple environments.

Drought adaptation in Arabidopsis thaliana by extensive genetic loss-of-function.

Interdisciplinary syntheses are needed to scale up discovery of the environmental drivers and molecular basis of adaptation in nature. Here we integrated novel approaches using whole genome sequences, satellite remote sensing, and transgenic experiments to study natural loss-of-function alleles associated with drought histories in wild Arabidopsis thaliana. The genes we identified exhibit population genetic signatures of parallel molecular evolution, selection for loss-of-function, and shared associations with flowering time phenotypes in directions consistent with longstanding adaptive hypotheses seven times more often than expected by chance. We then confirmed predicted phenotypes experimentally in transgenic knockout lines. These findings reveal the importance of drought timing to explain the evolution of alternative drought tolerance strategies and further challenge popular assumptions about the adaptive value of genetic loss-of-function in nature. These results also motivate improved species-wide sequencing efforts to better identify loss-of-function variants and inspire new opportunities for engineering climate resilience in crops.

Combining population genomics and fitness QTLs to identify the genetics of local adaptation in Arabidopsis thaliana.

Evidence for adaptation to different climates in the model species Arabidopsis thaliana is seen in reciprocal transplant experiments, but the genetic basis of this adaptation remains poorly understood. Field-based quantitative trait locus (QTL) studies provide direct but low-resolution evidence for the genetic basis of local adaptation. Using high-resolution population genomic approaches, we examine local adaptation along previously identified genetic trade-off (GT) and conditionally neutral (CN) QTLs for fitness between locally adapted Italian and Swedish A. thaliana populations [Ågren J, et al. (2013) Proc Natl Acad Sci USA 110:21077-21082]. We find that genomic regions enriched in high FST SNPs colocalize with GT QTL peaks. Many of these high FST regions also colocalize with regions enriched for SNPs significantly correlated to climate in Eurasia and evidence of recent selective sweeps in Sweden. Examining unfolded site frequency spectra across genes containing high FST SNPs suggests GTs may be due to more recent adaptation in Sweden than Italy. Finally, we collapse a list of thousands of genes spanning GT QTLs to 42 genes that likely underlie the observed GTs and explore potential biological processes driving these trade-offs, from protein phosphorylation, to seed dormancy and longevity. Our analyses link population genomic analyses and field-based QTL studies of local adaptation, and emphasize that GTs play an important role in the process of local adaptation.

TSPmap, a tool making use of traveling salesperson problem solvers in the efficient and accurate construction of high-density genetic linkage maps.

BACKGROUND: Recent advances in nucleic acid sequencing technologies have led to a dramatic increase in the number of markers available to generate genetic linkage maps. This increased marker density can be used to improve genome assemblies as well as add much needed resolution for loci controlling variation in ecologically and agriculturally important traits. However, traditional genetic map construction methods from these large marker datasets can be computationally prohibitive and highly error prone. RESULTS: We present TSPmap, a method which implements both approximate and exact Traveling Salesperson Problem solvers to generate linkage maps. We demonstrate that for datasets with large numbers of genomic markers (e.g. 10,000) and in multiple population types generated from inbred parents, TSPmap can rapidly produce high quality linkage maps with low sensitivity to missing and erroneous genotyping data compared to two other benchmark methods, JoinMap and MSTmap. TSPmap is open source and freely available as an R package. CONCLUSIONS: With the advancement of low cost sequencing technologies, the number of markers used in the generation of genetic maps is expected to continue to rise. TSPmap will be a useful tool to handle such large datasets into the future, quickly producing high quality maps using a large number of genomic markers.

Screening for Natural Variation in Water Use Efficiency Traits in a Diversity Set of Brassica napus L. Identifies Candidate Variants in Photosynthetic Assimilation.

Seed yield and quality of crop species are significantly reduced by water deficit. Stable isotope screening (δ13C) of a diversity set of 147 accessions of Brassica napus grown in the field identified several accessions with extremes in water use efficiency (WUE). We next conducted an investigation of the physiological characteristics of selected natural variants with high and low WUE to understand how these characteristics translate to differences in WUE. We identified an interesting Spring accession, G302 (Mozart), that exhibited the highest WUE in the field and high CO2 assimilation rates coupled with an increased electron transport capacity (Jmax) under the imposed conditions. Differences in stomatal density and stomatal index did not translate to differences in stomatal conductance in the investigated accessions. Stomatal conductance response to exogenous ABA was analyzed in selected high and low WUE accessions. Spring lines showed little variation in response to exogenous ABA, while one Semi-Winter line (SW047) showed a significantly more rapid response to exogenous ABA, that corresponded to the high WUE indicated by δ13C measurements. This research illustrates the importance of examining natural variation at a physiological level for investigation of the underlying mechanisms influencing the diversity of carbon isotope discrimination values in the field and identifies natural variants in B. napus with improved WUE and potential relevant traits.

Identification of Polymorphisms Associated with Drought Adaptation QTL in Brassica napus by Resequencing.

Brassica napus is a globally important oilseed for which little is known about the genetics of drought adaptation. We previously mapped twelve quantitative trait loci (QTL) underlying drought-related traits in a biparental mapping population created from a cross between winter and spring B. napus cultivars. Here we resequence the genomes of the mapping population parents to identify genetic diversity across the genome and within QTL regions. We sequenced each parental cultivar on the Illumina HiSeq platform to a minimum depth of 23 × and performed a reference based assembly in order to describe the molecular variation differentiating them at the scale of the genome, QTL and gene. Genome-wide patterns of variation were characterized by an overall higher single nucleotide polymorphism (SNP) density in the A genome and a higher ratio of nonsynonymous to synonymous substitutions in the C genome. Nonsynonymous substitutions were used to categorize gene ontology terms differentiating the parent genomes along with a list of putative functional variants contained within each QTL. Marker assays were developed for several of the discovered polymorphisms within a pleiotropic QTL on chromosome A10. QTL analysis with the new, denser map showed the most associated marker to be that developed from an insertion/deletion polymorphism located in the candidate gene Bna.FLC.A10, and it was the only candidate within the QTL interval with observed polymorphism. Together, these results provide a glimpse of genome-wide variation differentiating annual and biennial B. napus ecotypes as well as a better understanding of the genetic basis of root and drought phenotypes.

Exploiting Differential Gene Expression and Epistasis to Discover Candidate Genes for Drought-Associated QTLs in Arabidopsis thaliana.

Soil water availability represents one of the most important selective agents for plants in nature and the single greatest abiotic determinant of agricultural productivity, yet the genetic bases of drought acclimation responses remain poorly understood. Here, we developed a systems-genetic approach to characterize quantitative trait loci (QTLs), physiological traits and genes that affect responses to soil moisture deficit in the TSUxKAS mapping population of Arabidopsis thaliana. To determine the effects of candidate genes underlying QTLs, we analyzed gene expression as a covariate within the QTL model in an effort to mechanistically link markers, RNA expression, and the phenotype. This strategy produced ranked lists of candidate genes for several drought-associated traits, including water use efficiency, growth, abscisic acid concentration (ABA), and proline concentration. As a proof of concept, we recovered known causal loci for several QTLs. For other traits, including ABA, we identified novel loci not previously associated with drought. Furthermore, we documented natural variation at two key steps in proline metabolism and demonstrated that the mitochondrial genome differentially affects genomic QTLs to influence proline accumulation. These findings demonstrate that linking genome, transcriptome, and phenotype data holds great promise to extend the utility of genetic mapping, even when QTL effects are modest or complex.

Combining quantitative trait loci analysis with physiological models to predict genotype-specific transpiration rates.

Transpiration is controlled by evaporative demand and stomatal conductance (gs ), and there can be substantial genetic variation in gs . A key parameter in empirical models of transpiration is minimum stomatal conductance (g0 ), a trait that can be measured and has a large effect on gs and transpiration. In Arabidopsis thaliana, g0 exhibits both environmental and genetic variation, and quantitative trait loci (QTL) have been mapped. We used this information to create a genetically parameterized empirical model to predict transpiration of genotypes. For the parental lines, this worked well. However, in a recombinant inbred population, the predictions proved less accurate. When based only upon their genotype at a single g0 QTL, genotypes were less distinct than our model predicted. Follow-up experiments indicated that both genotype by environment interaction and a polygenic inheritance complicate the application of genetic effects into physiological models. The use of ecophysiological or 'crop' models for predicting transpiration of novel genetic lines will benefit from incorporating further knowledge of the genetic control and degree of independence of core traits/parameters underlying gs variation.

QTL analysis of root morphology, flowering time, and yield reveals trade-offs in response to drought in Brassica napus.

Drought escape and dehydration avoidance represent alternative strategies for drought adaptation in annual crops. The mechanisms underlying these two strategies are reported to have a negative correlation, suggesting a trade-off. We conducted a quantitative trait locus (QTL) analysis of flowering time and root mass, traits representing each strategy, in Brassica napus to understand if a trade-off exists and what the genetic basis might be. Our field experiment used a genotyped population of doubled haploid lines and included both irrigated and rainfed treatments, allowing analysis of plasticity in each trait. We found strong genetic correlations among all traits, suggesting a trade-off among traits may exist. Summing across traits and treatments we found 20 QTLs, but many of these co-localized to two major QTLs, providing evidence that the trade-off is genetically constrained. To understand the mechanistic relationship between root mass, flowering time, and QTLs, we analysed the data by conditioning upon correlated traits. Our results suggest a causal model where such QTLs affect root mass directly as well as through their impacts on flowering time. Additionally, we used draft Brassica genomes to identify orthologues of well characterized Arabidopsis thaliana flowering time genes as candidate genes. This research provides valuable clues to breeding for drought adaptation as it is the first to analyse the inheritance of the root system in B. napus in relation to drought.

Development of a next-generation NIL library in Arabidopsis thaliana for dissecting complex traits.

BACKGROUND: The identification of the loci and specific alleles underlying variation in quantitative traits is an important goal for evolutionary biologists and breeders. Despite major advancements in genomics technology, moving from QTL to causal alleles remains a major challenge in genetics research. Near-isogenic lines are the ideal raw material for QTL validation, refinement of QTL location and, ultimately, gene discovery. RESULTS: In this study, a population of 75 Arabidopsis thaliana near-isogenic lines was developed from an existing recombinant inbred line (RIL) population derived from a cross between physiologically divergent accessions Kas-1 and Tsu-1. First, a novel algorithm was developed to utilize genome-wide marker data in selecting RILs fully isogenic to Kas-1 for a single chromosome. Seven such RILs were used in 2 generations of crossing to Tsu-1 to create BC1 seed. BC1 plants were genotyped with SSR markers so that lines could be selected that carried Kas-1 introgressions, resulting in a population carrying chromosomal introgressions spanning the genome. BC1 lines were genotyped with 48 genome-wide SSRs to identify lines with a targeted Kas-1 introgression and the fewest genomic introgressions elsewhere. 75 such lines were selected and genotyped at an additional 41 SNP loci and another 930 tags using 2b-RAD genotyping by sequencing. The final population carried an average of 1.35 homozygous and 2.49 heterozygous introgressions per line with average introgression sizes of 5.32 and 5.16 Mb, respectively. In a simple case study, we demonstrate the advantage of maintaining heterozygotes in our library whereby fine-mapping efforts are conducted simply by self-pollination. Crossovers in the heterozygous interval during this single selfing generation break the introgression into smaller, homozygous fragments (sub-NILs). Additionally, we utilize a homozygous NIL for validation of a QTL underlying stomatal conductance, a low heritability trait. CONCLUSIONS: The present results introduce a new and valuable resource to the Brassicaceae research community that enables rapid fine-mapping of candidate loci in parallel with QTL validation. These attributes along with dense marker coverage and genome-wide chromosomal introgressions make this population an ideal starting point for discovery of genes underlying important complex traits of agricultural and ecological significance.

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.

Shade avoidance and the regulation of leaf inclination in Arabidopsis.

As a rosette plant, Arabidopsis thaliana forms leaves near to the ground, which causes the plant to be vulnerable to shading by neighbours. One mechanism to avoid such shading is the regulation of leaf inclination, such that leaves can be raised to more vertical orientations to prevent neighbouring leaves from overtopping them. Throughout Arabidopsis rosette development, rosette leaves move to more vertical orientations when shaded by neighbouring leaves, exposed to low light levels or placed in the dark. After dark-induced reorientation of leaves, returning them to white light causes the leaves to reorient to more horizontal inclinations. These light-dependent leaf movements are more robust than, and distinct from, the diurnal movements of rosette leaves. However, the movements are gated by the circadian clock. The light-dependent leaf orientation response is mediated primarily through phytochromes A, B and E, with the orientation varying with the ratio of red light to far-red light, consistent with other shade-avoidance responses. However, even plants lacking these phytochromes were able to alter leaf inclination in response to white light, suggesting a role for other photoreceptors. In particular, we found significant changes in leaf inclination for plants exposed to green light. This green light response may be caused, in part, by light-dependent regulation of abscisic acid (ABA) biosynthesis.

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.

Phytochrome modulation of blue light-induced chloroplast movements in Arabidopsis.

Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 micromol m-2 s-1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5-15 micromol m-2 s-1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.

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.

Root gravitropism in response to a signal originating outside of the cap.

We have developed image analysis software linked to a rotating stage, allowing constraint of any user-selected region of a root at a prescribed angle during root gravitropism. This device allows the cap of a graviresponding root to reach vertical while maintaining a selected region within the elongation zone at a gravistimulated angle. Under these conditions gravitropic curvature of roots of Zea mays L. continues long after the root cap reaches vertical, indicating that a signal from outside of the cap can contribute to the curvature response.