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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.

The root system is critical for the survival of nearly all land plants and a key target for improving abiotic stress tolerance, nutrient accumulation, and yield in crop species. Although many methods of root phenotyping exist, within field studies, one of the most popular methods is the extraction and measurement of the upper portion of the root system, known as the root crown, followed by trait quantification based on manual measurements or 2D imaging. However, 2D techniques are inherently limited by the information available from single points of view. Here, we used X-ray computed tomography to generate highly accurate 3D models of maize root crowns and created computational pipelines capable of measuring 71 features from each sample. This approach improves estimates of the genetic contribution to root system architecture and is refined enough to detect various changes in global root system architecture over developmental time as well as more subtle changes in root distributions as a result of environmental differences. We demonstrate that root pulling force, a high-throughput method of root extraction that provides an estimate of root mass, is associated with multiple 3D traits from our pipeline. Our combined methodology can therefore be used to calibrate and interpret root pulling force measurements across a range of experimental contexts or scaled up as a stand-alone approach in large genetic studies of root system architecture.

Understanding how genomic and expression variation is linked to adaptation of plants to local environments is fundamental to the fields of evolutionary biology and species conservation. Using locally adaptedArabidopsis thalianaItaly and Sweden populations, we examine how variation in gene expression under control and cold acclimation conditions, is linked to allele frequency differentiation (AFD); linkage disequilibrium (LD); selective constraint at nonsynonymous sites; and genetic-tradeoff quantitative trait loci (GT-QTL). Our results indicate that contrary to genes showing a main effect in environment (E), expression genotype by environment interactions (GxE) show significantly higher AFD along cis-regulatory and nonsynonymous sites than the neutral expectation; and interestingly, highly differentiated GxE genes show higher expression and inter-species selective constraint than the rest of the genes. When examining the association between genomic signatures of selection along GxE/E genes and GT-QTL, we find that GxE genes showing a high AFD and LD, display a significant and much higher enrichment along GT-QTL than the genome-wide/E set of genes. Nonetheless, E genes show a higher enrichment than the genome-wide control. In summary, our results suggest, that these highly expressed and selectively constrained GxE genes, may have been part of a cold-responsive regulon of E genes that experienced recent selection when migrating to new environments. Candidate GxE genes underlying GT-QTL reveal interesting biological processes that may underlie local adaptation to temperature, including flowering time, light-dependent cold acclimation, freezing tolerance, and response to hypoxia. Finally, we find no evidence linking lower expression of the CBF-dependent freezing tolerance pathway to genetic-tradeoffs and adaptation to warmer climates.

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.

Carbon isotope ratio (δ13C) and root pulling force (RPF) are associated with dehydration avoidance in plants. The δ13C measures the efficiency of C gain relative to water loss, whereas RPF, the vertical force required to pull plants from the ground, indirectly estimates root‐related dehydration avoidance capacity. To determine quantitative trait loci (QTL) controlling these traits in canola (Brassica napus L.), a mapping population was evaluated in 2011 and 2012 in Fort Collins, CO. The split‐plot experiment had two moisture regimes (main plot) and 148 doubled haploid lines (subplot), replicated three times. Analysis of variance indicated highly significant (P

Lateral roots of many species are maintained at non-vertical angles with respect to gravity. These gravitropic setpoint angles (GSAs) are intriguing because their maintenance requires that roots are able to effect gravitropic response both with and against the gravity vector. Here we have used the Arabidopsis lateral root in order to investigate the molecular basis of the maintenance of non-vertical GSAs. We show that gravitropism in the lateral root is angle-dependent and that both upward and downward graviresponse requires auxin transport and the generation of auxin asymmetries consistent with the Cholodny-Went model. We show that the symmetry in auxin distribution in lateral roots growing at GSA can be traced back to a net, balanced polarization of PIN3 and PIN7 auxin transporters in the columella cells. Further, upward and downward graviresponse in lateral roots correlates with corresponding changes in PIN3 and PIN7 polar localisation. Finally, we show that auxin, in addition to driving tropic growth in the lateral root, acts within the columella to regulate GSA via the PIN phosphatase subunit RCN1 in a PIN3-dependent and PIN7-independent manner. Together, these findings provide a molecular framework for understanding gravity-dependent nonvertical growth in Arabidopsis lateral roots.

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., eLife digest Water shortages caused by droughts lead to crop losses that affect billions of people around the world each year. By discovering how wild plants adapt to drought, it may be possible to identify traits and genes that help to improve the growth of crop plants when water is scarce. It has been suggested that plants have adapted to droughts by flowering at times of the year when droughts are less likely to occur. For example, if droughts are more likely to happen in spring, the plants may delay flowering until the summer. Arabidopsis thaliana is a small plant that is found across Eurasia, Africa and North America, including in areas that are prone to drought at different times of the year. Individual plants of the same species may carry different versions of the same gene (known as alleles). Some of these alleles may not work properly and are referred to as loss-of-function alleles. Monroe et al. investigated whether A. thaliana plants carry any loss-of-function alleles that are associated with droughts happening in the spring or summer, and whether they are linked to when those plants will flower. Monroe et al. analyzed satellite images collected over the last 30 years to measure when droughts have occurred. Next, they searched genome sequences of Arabidopsis thaliana for alleles that might help the plants to adapt to droughts in the spring or summer. Combining the two approaches revealed that loss-of-function alleles associated with spring droughts were strongly predicted to be associated with the plants flowering later in the year. Similarly, loss-of-function alleles associated with summer droughts were predicted to be associated with the plants flowering earlier in the year. These findings support the idea that plants can adapt to drought by changing when they produce flowers, and suggest that loss-of-function alleles play a major role in this process. New techniques for editing genes mean it is easier than ever to generate new loss-of-function alleles in specific genes. Therefore, the results presented by Monroe et al. may help researchers to develop new varieties of crop plants that are better adapted to droughts.

Visions of a second green revolution empowered by emerging technologies have called for interdisciplinary syntheses to scale up the discovery of functionally definitive gene variants responsible for climate adaptation in plants. We integrated novel approaches using whole genome sequences and satellite remote sensing to identify natural knockout alleles associated with drought histories in wild Arabidopsis thaliana. Genes identified exhibit signatures of parallel molecular evolution, selection for loss-of-function alleles, and shared associations with flowering time phenotypes in directions consistent with longstanding adaptive hypotheses 7 times more often than expected by chance. Artificial knockout lines then confirmed predicted phenotypes experimentally. These findings further challenge popular assumptions about the adaptive value of genetic loss-of-function in nature and inspire new opportunities for engineering climate resilience in crops.One sentence summaryWhole genome sequences and satellite-detected droughts point to gene knockouts as valuable genetic fuel for climate adaptation.

1169 I. 1170 II. 1170 III. 1172 IV. 1176 V. 1181 VI. 1182 1183 References 1183 SUMMARY: Modern agriculture is facing multiple challenges including the necessity for a substantial increase in production to meet the needs of a burgeoning human population. Water shortage is a deleterious consequence of both population growth and climate change and is one of the most severe factors limiting global crop productivity. Brassica species, particularly canola varieties, are cultivated worldwide for edible oil, animal feed, and biodiesel, and suffer dramatic yield loss upon drought stress. The recent release of the Brassica napus genome supplies essential genetic information to facilitate identification of drought-related genes and provides new information for agricultural improvement in this species. Here we summarize current knowledge regarding drought responses of canola, including physiological and -omics effects of drought. We further discuss knowledge gained through translational biology based on discoveries in the closely related reference species Arabidopsis thaliana and through genetic strategies such as genome-wide association studies and analysis of natural variation. Knowledge of drought tolerance/resistance responses in canola together with research outcomes arising from new technologies and methodologies will inform novel strategies for improvement of drought tolerance and yield in this and other important crop species.

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.

Significance Local adaptation can occur due to individual genetic variants that increase the fitness of individuals in their home environments but decrease fitness in other environments [genetic trade-offs (GTs)] or genetic variants that increase fitness in one environment but have no effect in other environments [conditional neutrality (CN)]. Here, we show that GT quantitative trait loci (QTLs) for fitness between Italian and Swedish Arabidopsis thaliana exhibit strong population genomic signatures of local adaptation, including elevated levels of allele frequency differentiation, correlations to climatic variables, and recent sweeps. Highly divergent genes between Italy and Sweden populations show evidence of more recent selection in Sweden than Italy, and the biological annotations of these genes suggest interesting mechanisms underlying local adaptation., 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.

Highlight text This research is the first to examine the genetics of drought adaptive traits in Brassica napus and is supportive of an inherent trade-off between resource investment in reproduction and root development., 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.

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.

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.

Identifying the genetic basis of adaptation to climate has long been a goal in evolutionary biology and has applications in agriculture. Adaptation to drought represents one important aspect of local adaptation, and drought is the major factor limiting agricultural yield. We examined local adaptation between Sweden and Italy Arabidopsis thaliana ecotypes, which show contrasting levels of water availability in their local environments. To identify quantitative trait loci (QTL) controlling water use physiology traits and adaptive trait QTL (genomic regions where trait QTL and fitness QTL colocalize), we performed QTL mapping on 374 F 9 recombinant inbred lines in well-watered and terminal drought conditions. We found 72 QTL (32 in well-watered, 31 in drought, 9 for plasticity) across five water use physiology traits: δ 13 C, rosette area, dry rosette weight, leaf water content and percent leaf nitrogen. Some of these genomic regions colocalize with fitness QTL and with other physiology QTL in defined hotspots. In addition, we found evidence of both constitutive and inducible water use physiology QTL. Finally, we identified highly divergent candidate genes, in silico. Our results suggest that many genes with minor effects may influence adaptation through water use physiology and that pleiotropic water use physiology QTL have fitness consequences.

Phototropism and gravitropism play key roles 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. The blue-light response is controlled by the phototropins while the red-light response is mediated by the phytochrome family of photoreceptors. In order to better characterize root phototropism, we plan to perform experiments in microgravity so that this tropism can be more effectively studied without the interactions with the gravity response. Our experiments are to be performed on the European Modular Cultivation System (EMCS), which provides an incubator, lighting system, and high resolution video that are on a centrifuge palette. These experiments will be performed at μ g , 1 g (control) and fractional g -levels. In order to ensure success of this mission on the International Space Station, we have been conducting ground-based studies on growth, phototropism, and gravitropism in experimental unique equipment (EUE) that was designed for our experiments with Arabidopsis seedlings. Currently, the EMCS and our EUE are scheduled for launch on space shuttle mission STS-121. This project should provide insight into how the blue- and red-light signaling systems interact with each other and with the gravisensing system.

Most research on gravity responses in plants has focused on primary roots and shoots, which typically grow in a vertical orientation. However, the patterns of lateral organ growth, which generally have large effects on overall plant architecture, are such that the organs are typically not vertical. In lateral roots of Arabidopsis, growth is initially in a nearly horizontal orientation but changes to a near-vertical orientation as the lateral root develops. Although the non-vertical lateral roots are gravitropically competent, following gravitropic reorientation of seedlings, the lateral roots on the upper flank of the primary root have different growth patterns from those on the lower side of the primary root. The differences are in part dependent on reorientation of the primary root, suggesting that gravitropic signaling from the primary root also contributes to the control of lateral root growth. The hormone auxin appears to play a role in this signaling between the primary and lateral roots, as auxin transport inhibitors applied to the primary root affect lateral root growth. Also, lateral roots of pin3 mutants, which are impaired in polar auxin transport, have altered lateral root orientations. However, other signals from the primary root tip also play an important role in regulating lateral root growth.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Although the effects of gravity on root growth are well known and interactions between light and gravity have been reported, details of root phototropic responses are less documented. We used high-resolution image analysis to study phototropism in primary roots of Zea mays L. Similar to the location of perception in gravitropism, the perception of light was localized in the root cap. Phototropic curvature away from the light, on the other hand, developed in the central elongation zone, more basal than the site of initiation of gravitropic curvature. The phototropic curvature saturated at approximately 10 micromoles m-2 s-1 blue light with a peak curvature of 29 +/- 4 degrees, in part due to induction of positive gravitropism following displacement of the root tip from vertical during negative phototropism. However, at higher fluence rates, development of phototropic curvature is arrested even if gravitropism is avoided by maintaining the root cap vertically using a rotating feedback system. Thus continuous illumination can cause adaptation in the signalling pathway of the phototropic response in roots.

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.

Although exogenous electric fields have been reported to influence the orientation of plant root growth, reports of the ultimate direction of differential growth have been contradictory. Using a high-resolution image analysis approach, the kinetics of electrotropic curvature in Vigna mungo L. roots were investigated. It was found that curvature occurred in the same root toward both the anode and cathode. However, these two responses occurred in two different regions of the root, the central elongation zone (CEZ) and distal elongation zone (DEZ), respectively. These oppositely directed responses could be reproduced individually by a localized electric field application to the region of response. This indicates that both are true responses to the electric field, rather than one being a secondary response to an induced gravitropic stimulation. The individual responses differed in the type of differential growth giving rise to curvature. In the CEZ, curvature was driven by inhibition of elongation, whereas curvature in the DEZ was primarily due to stimulation of elongation. This stimulation of elongation is consistent with the growth response of the DEZ to other environmental stimuli.

In this study we investigated the kinetics of the gravitropic response of the Arabidopsis mutant rgr1 (reducedroot gravitropism). Although the rate of curvature in rgr1, which is allelic toaxr4, was smaller than in the wild type (ecotype Wassilewskija), curvature was initiated in the same region of the root, the distal elongation zone. The time lag for the response was unaffected in the mutant; however, the gravitropic response ofrgr1 contained a feature not found in the wild type: when roots growing along the surface of an agar plate were gravistimulated, there was often an upward curvature that initiated in the central elongation zone. Because this response was dependent on the tactile environment of the root, it most likely resulted from the superposition of the waving/coiling phenomenon onto the gravitropic response. We found that the frequency of the waving pattern and circumnutation, a cyclic endogenous pattern of root growth, was the same in rgr1 and in the wild type, so the waving/coiling phenomenon is likely governed by circumnutation patterns. The amplitudes of these oscillations may then be selectively amplified by tactile stimulation to provide a directional preference to the slanting.

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.

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.

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.

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.

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 μmol 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 μmol 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.

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.

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° to 40°, compared with 5° to 10° 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 andphyB were severely impaired in red-light-induced phototropism, whereas the phyD and phyEmutants 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.

Roots grown in an applied electric field demonstrate a bidirectional curvature. To further understand the nature of this response and its implications for the regulation of differential growth, we applied an electric field to roots growing in microgravity. We found that growth rates of roots in microgravity were higher than growth rates of ground controls. Immediately upon application of the electric field, root elongation was inhibited. We interpret this result as an indication that, in the absence of a gravity stimulus, the sensitivity of the root to an applied electric stimulus is increased. Further space experiments are required to determine the extent to which this sensitivity is shifted. The implications of this result are discussed in relation to gravitropic signaling and the regulation of differential cell elongation in the root.

The study of gravitropism is hindered by the fact that as a root responds, the gravitational stimulus changes. Using a feedback system to connect a rotating stage platform to a video digitizer system, we were able to maintain a constant angle of gravistimulation to Arabidopsis roots for long time periods. The rate of curvature approximated the sine rule for angles of stimulation between 20° and 120°. For a given angle of stimulation, the rate of curvature also remained constant, with no observed diminishment of the response. Although previous reports of Arabidopsis root gravitropism suggest latent periods of approximately 30 min, using a smooth mechanical stage to reorient the root, we observed a mean time lag of approximately 10 min. This more rapid onset of curvature can, in part, be explained by reduced mechanical perturbation during the process of gravistimulation. This suggests that mechanical stimulation associated with rapid root re-orientation may confound investigations of early gravitropic events.

Although Arabidopsis is an important system for studying root physiology, the localized growth patterns of its roots have not been well defined, particularly during tropic responses. In order to characterize growth rate profiles along the apex of primary roots of Arabidopsis thaliana (L.) Heynh (ecotype Columbia) we applied small charcoal particles to the root surface and analyzed their displacement during growth using an automated video digitizer system with custom software for tracking the markers. When growing vertically, the maximum elongation rate occurred 481 +/- 50 microns back from the extreme tip of the root (tip of root cap), and the elongation zone extended back to 912 +/- 137 microns. The distal elongation zone (DEZ) has previously been described as the apical region of the elongation zone in which the relative elemental growth rate (REGR) is < or = 30% of the peak rate in the central elongation zone. By this definition, our data indicate that the basal limit of the DEZ was located 248 +/- 30 microns from the root tip. However, after gravistimulation, the growth patterns of the root changed. Within the first hour of graviresponse, the basal limit of the DEZ and the position of peak REGR shifted apically on the upper flank of the root. This was due to a combination of increased growth in the DEZ and growth inhibition in the central elongation zone. On the lower flank, the basal limit of the DEZ shifted basipetally as the REGR decreased. These factors set up the gradient of growth rate across the root, which drives curvature.