Structure and genetic diversity of Canadian Maritimes wild hops.

Studies on the northeastern American native hops (Humulus lupulus ssp. lupuloides) from the Canadian Maritimes are scarce. This study aimed to evaluate the genetic structure and diversity among 25 wild-collected hops from three Canadian Maritime provinces using microsatellite (simple sequence repeat (SSR)) markers. Based on 43 SSR markers, four distinct subgroups were found, with a low molecular variance (19%) between subgroups and a high variance (81%) within subgroups. The Nei's unbiased genetic distance between clusters ranged from 0.01 to 0.08, the genetic distance between clusters 2 and 3 being the farthest and that between clusters 1 and 2 the closest. Cluster 2 captured the highest overall diversity. A total of 18 SSR markers clearly discriminated hop clones by detecting putative subspecies-specific haplotypes, differentiating clones of native-wild H. lupulus ssp. lupuloides from the naturalized old and modern hop cultivars. Seven of the 18 SSR markers also differentiated two clones from the same site from one another. The study is the first, using molecular markers, to identify SSR markers with potential for intellectual property protection in Canadian Maritimes hops. The SSR markers herein used can be prime tools for hop breeders and growers in the region.

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known.

Comparative transcriptome expression analysis in susceptible and resistant potato (Solanum tuberosum) cultivars to common scab (Streptomyces scabies) revealed immune priming responses in the incompatible interaction.

Common scab disease in potato has become a widespread issue in major potato production areas, leading to increasing economic losses. Varietal resistance is seen as a viable and long-term scab management strategy. However, the genes and mechanisms of varietal resistance are unknown. In the current study, a comparative RNA transcriptome sequencing and differential gene signaling and priming sensitization studies were conducted in two potato cultivars that differ by their response to common scab (Streptomyces scabies), for unraveling the genes and pathways potentially involved in resistance within this pathosystem. We report on a consistent and contrasted gene expression pattern from 1,064 annotated genes differentiating a resistant (Hindenburg) and a susceptible (Green Mountain) cultivars, and identified a set of 273 co-regulated differentially expressed genes in 34 pathways that more likely reflect the genetic differences of the cultivars and metabolic mechanisms involved in the scab pathogenesis and resistance. The data suggest that comparative transcriptomic phenotyping can be used to predict scab lesion phenotype in breeding lines using mature potato tuber. The study also showed that the resistant cultivar, Hindenburg, has developed and maintained a capacity to sense and prime itself for persistent response to scab disease over time, and suggests an immune priming reaction as a mechanism for induced-resistance in scab resistant potato cultivars. The set of genes identified, described, and discussed in the study paves the foundation for detailed characterizations towards tailoring and designing procedures for targeted gene knockout through gene editing and phenotypic evaluation.

Fast and accurate genomic analyses using genome graphs.

The human reference genome serves as the foundation for genomics by providing a scaffold for alignment of sequencing reads, but currently only reflects a single consensus haplotype, thus impairing analysis accuracy. Here we present a graph reference genome implementation that enables read alignment across 2,800 diploid genomes encompassing 12.6 million SNPs and 4.0 million insertions and deletions (indels). The pipeline processes one whole-genome sequencing sample in 6.5 h using a system with 36 CPU cores. We show that using a graph genome reference improves read mapping sensitivity and produces a 0.5% increase in variant calling recall, with unaffected specificity. Structural variations incorporated into a graph genome can be genotyped accurately under a unified framework. Finally, we show that iterative augmentation of graph genomes yields incremental gains in variant calling accuracy. Our implementation is an important advance toward fulfilling the promise of graph genomes to radically enhance the scalability and accuracy of genomic analyses.

Electrical Microstimulation of Visual Cerebral Cortex Elevates Psychophysical Detection Thresholds.

Sensory prostheses can restore aspects of natural sensation by delivering electrical current directly into sensory circuits. An effective sensory prosthetic should be capable of generating reliable real-time perceptual signals for hours each day over many years. However, we still know little regarding the stability of percepts produced by electrical microstimulation of cerebral sensory cortex when stimulation is delivered repeatedly over long periods. Developing methods that yield highly sensitive and reliable assessments of a subject's sensitivity to stimulation is important for developing prosthetic devices that can mimic the constant stream of information inherent in daily experience. Here, we trained rhesus monkeys to report electrical microstimulation of their primary visual cortex (V1) and measured how repeated stimulation affected the minimal electrical current needed to generate a percept (behavioral detection threshold). Using adaptive staircase procedures with a two-alternative forced-choice (2AFC) detection task, we obtained highly reliable detection threshold measures with as few as 100 trials. Using either chronically implanted or acutely inserted microelectrodes, we found that repeated electrical microstimulation elevated detection thresholds, with effects persisting between daily testing sessions. Our results demonstrate task designs that can support rapid and reliable measurements of detection thresholds, and point to the need for validation that detection thresholds in targeted structures will be sufficiently stable in the face of the amount of chronic stimulation that will be required for effective sensory prosthetics.

Induced Mutagenesis in UGT74S1 Gene Leads to Stable New Flax Lines with Altered Secoisolariciresinol Diglucoside (SDG) Profiles.

Flax secoisolariciresinol (SECO) diglucoside (SDG) lignan is an emerging natural product purported to prevent chronic diseases in humans. SECO, the aglycone form of SDG, has shown higher intestinal cell absorption but it is not accumulated naturally in planta. Recently, we have identified and characterized a UDP-glucosyltransferase gene, UGT74S1, that glucosylates SECO into its monoglucoside (SMG) and SDG forms when expressed in yeast. However, whether this gene is unique in controlling SECO glucosylation into SDG in planta is unclear. Here, we report on the use of UGT74S1 in reverse and forward genetics to characterize an ethyl methane sulfonate (EMS) mutagenized flax population from cultivar CDC Bethune and consisting of 1996 M2 families. EMS mutagenesis generated 73 SNP variants causing 79 mutational events in the UGT74S1 exonic regions of 93 M2 families. The mutation frequency in the exonic regions was determined to be one per 28 Kb. Of these mutations, 13 homozygous missense mutations and two homozygous nonsense mutations were observed and all were transmitted into the M3 and M4 generations. Forward genetics screening of the population showed homozygous nonsense mutants completely lacking SDG biosynthesis while the production of SMG was observed only in a subset of the M4 lines. Heterozygous or homozygous M4 missense mutants displayed a wide range of SDG levels, some being greater than those of CDC Bethune. No additional deleterious mutations were detected in these mutant lines using a panel of 10 other genes potentially involved in the lignan biosynthesis. This study provides further evidence that UGT74S1 is unique in controlling SDG formation from SECO and this is the first report of non-transgenic flax germplasm with simultaneous knockout of SDG and presence of SMG in planta.

UGT74S1 is the key player in controlling secoisolariciresinol diglucoside (SDG) formation in flax.

BACKGROUND: Flax lignan, commonly known as secoisolariciresinol (SECO) diglucoside (SDG), has recently been reported with health-promoting activities, including its positive impact in metabolic diseases. However, not much was reported on the biosynthesis of SDG and its monoglucoside (SMG) until lately. Flax UGT74S1 was recently reported to sequentially glucosylate SECO into SMG and SDG in vitro. However, whether this gene is the only UGT achieving SECO glucosylation in flax was not known. RESULTS: Flax genome-wide mining for UGTs was performed. Phylogenetic and gene duplication analyses, heterologous gene expression and enzyme assays were conducted to identify family members closely related to UGT74S1 and to establish their roles in SECO glucosylation. A total of 299 different UGTs were identified, of which 241 (81%) were duplicated. Flax UGTs diverged 2.4-153.6 MYA and 71% were found to be under purifying selection pressure. UGT74S1, a single copy gene located on chromosome 7, displayed no evidence of duplication and was deemed to be under positive selection pressure. The phylogenetic analysis identified four main clusters where cluster 4, which included UGT74S1, was the most diverse. The duplicated UGT74S4 and UGT74S3, located on chromosomes 8 and 14, respectively, were the most closely related to UGT74S1 and were differentially expressed in different tissues. Heterologous expression levels of UGT74S1, UGT74S4 and UGT74S3 proteins were similar but UGT74S4 and UGT74S3 glucosylation activity towards SECO was seven fold less than UGT74S1. In addition, they both failed to produce SDG, suggesting neofunctionalization following their divergence from UGT74S1. CONCLUSIONS: We showed that UGT74S1 is closely related to two duplicated genes, UGT74S4 and UGT74S3 which, unlike UGT74S1, failed to glucosylate SMG into SDG. The study suggests that UGT74S1 may be the key player in controlling SECO glucosylation into SDG in flax although its closely related genes may also contribute to a minor extent in supplying the SMG precursor to UGT74S1.

Histidine 352 (His352) and tryptophan 355 (Trp355) are essential for flax UGT74S1 glucosylation activity toward secoisolariciresinol.

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser357 and Trp355 as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys335, Gln337 and Trp355 were predicted to bind the 7-OH, 2-OCH3 and 17-OCH3 of SECO. Site-directed mutagenesis of Cys335, Gln337, His352, Trp355 and Ser357, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp355 was substituted to Ala355 and Gly355 or when changing His352 to Asp352, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp355 and His352 are critical for UGT74S1's glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

BACKGROUND: Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. RESULTS: Five UGT genes belonging to the glycosyltransferases' family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. CONCLUSION: We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

An open source 3-d printed modular micro-drive system for acute neurophysiology.

Current, commercial, electrode micro-drives that allow independent positioning of multiple electrodes are expensive. Custom designed solutions developed by individual laboratories require fabrication by experienced machinists working in well equipped machine shops and are therefore difficult to disseminate into widespread use. Here, we present an easy to assemble modular micro-drive system for acute primate neurophysiology (PriED) that utilizes rapid prototyping (3-d printing) and readily available off the shelf-parts. The use of 3-d printed parts drastically reduces the cost of the device, making it available to labs without the resources of sophisticated machine shops. The direct transfer of designs from electronic files to physical parts also gives researchers opportunities to easily modify and implement custom solutions to specific recording needs. We also demonstrate a novel model of data sharing for the scientific community: a publicly available repository of drive designs. Researchers can download the drive part designs from the repository, print, assemble and then use the drives. Importantly, users can upload their modified designs with annotations making them easily available for others to use.

A strong constraint to the joint processing of pairs of cortical signals.

An important question in neuroscience is how the activity from spatially distributed cortical representations is integrated and processed together. In this study, we used a new approach to investigate the integration of distributed cortical activity. We used microstimulation to directly activate pairs of sites in primary visual cortex of rhesus monkeys. The sites were activated either singly or jointly, and the monkeys were trained to behaviorally report detection of the activation of either cortical site. We compared the detection performance with predictions from two different mathematical models of signal combination. Our data show that, at cortical separations <1 mm, signal integration is well described as a linear combination (d' summation) of individual site activity. At larger separations, signal integration is better described as a maximum operation on the site signals. We compare our neurophysiological findings to existing psychophysical data and suggest the intriguing possibility that cortical activity originating at spatial separations greater than ∼1 mm is processed as if by parallel, independent circuits whose signals can be compared against each other but not summed. This in turn implies that there is a strong constraint to the kinds of computations the brain can perform with spatially distributed cortical activity.

Acoustic scanning of natural scenes by echolocation in the big brown bat, Eptesicus fuscus.

Echolocation allows bats to orient and localize prey in complete darkness. The sonar beam of the big brown bat, Eptesicus fuscus, is directional but broad enough to provide audible echo information from within a 60-90 deg. cone. This suggests that the big brown bat could interrogate a natural scene without fixating each important object separately. We tested this idea by measuring the directional aim and duration of the bat's sonar beam as it performed in a dual task, obstacle avoidance and insect capture. Bats were trained to fly through one of two openings in a fine net to take a tethered insect at variable distances behind the net. The bats sequentially scanned the edges of the net opening and the prey by centering the axis of their sonar beam with an accuracy of approximately 5 deg. The bats also shifted the duration of their sonar calls, revealing sequential sampling along the range axis. Changes in duration and directional aim were correlated, showing that the bat first inspected the hole, and then shifted its gaze to the more distant insect, before flying through the net opening. Contrary to expectation based on the sonar beam width, big brown bats encountering a complex environment accurately pointed and shifted their sonar gaze to sequentially inspect closely spaced objects in a manner similar to visual animals using saccades and fixations to scan a scene. The findings presented here from a specialized orientation system, echolocation, offer insights into general principles of active sensing across sensory modalities for the perception of natural scenes.

Behavioral responses of big brown bats to dives by praying mantises.

Insectivorous echolocating bats face a formidable array of defenses employed by their airborne prey. One such insect defense is the ultrasound-triggered dive, which is a sudden, rapid drop in altitude, sometimes all the way to the ground. Although many previous studies have investigated the dynamics of such dives and their effect on insect survival rate, there has been little work on how bats may adapt to such an insect defense employed in the middle of pursuit. In this study we investigated how big brown bats (Eptesicus fuscus) adjust their pursuit strategy when flying praying mantises (Parasphendale agrionina) execute evasive, ultrasound-triggered dives. Although the mantis dive occasionally forced the bat to completely abort its chase (25% trials), in a number of cases (75% trials) the bat followed the mantis into the dive. In such cases the bat kept its sonar beam locked onto the target and maneuvered to maintain the same time efficient strategy it adopted during level flight pursuit, though it was ultimately defeated by the dive. This study suggests that although the mantis dive can be effective in evading the bat, it does not always deter the bat from continuing pursuit and, given enough altitude, the bat can potentially capture diving prey using the same flight strategy it employs to intercept prey in level flight.

Differential profiling of selected defence-related genes induced on challenge with Alternaria brassicicola in resistant white mustard and their comparative expression pattern in susceptible India mustard.

The lack of availability of sources of resistance against Alternaria brassicicola within the family Brassicaceae has made oilseed mustard plants a target for one of the most damaging and widespread fungal diseases, Alternaria black spot. Of the other non-host-resistant/tolerant plants, Sinapis alba, white mustard, is considered to be the most important apart from Arabidopsis. To understand the defence response of S. alba upon incompatible interaction with this pathogen, a functional genomic approach using cDNA amplified fragment length polymorphism was performed. The highly reproducible bands, found to be either more amplified or uniquely present in infected S. alba plants compared with non-infected plants, were further subjected to comparative reverse Northern analysis in the incompatible white mustard (S. alba) and compatible India mustard (Brassica juncea L.) plants. The suppression of 46% of the genes in the compatible background indicates the possibility of effective and specific recognition of Alternaria in S. alba. Analysis of the 118 genes up-regulated specifically in infected S. alba compared with B. juncea showed that 98 genes have similarity to proteins such as receptor-like protein kinase genes, genes involved with calcium-mediated signalling and salicylic acid-dependent genes as well as other genes of known function in Arabidopsis. The apparent expression profile data were further confirmed for selected genes by quantitative real-time polymerase chain reaction analysis. Classification of these genes on the basis of their induction pattern in Arabidopsis indicates that the expression profile of several of these genes was distinct in S. alba compared with B. juncea.

Flying big brown bats emit a beam with two lobes in the vertical plane.

The sonar beam of an echolocating bat forms a spatial window restricting the echo information returned from the environment. Investigating the shape and orientation of the sonar beam produced by a bat as it flies and performs various behavioral tasks may yield insight into the operation of its sonar system. This paper presents recordings of vertical and horizontal cross sections of the sonar beam produced by Eptesicus fuscus (big brown bats) as they fly and pursue prey in a laboratory flight room. In the horizontal plane the sonar beam consists of one large lobe and in the vertical plane the beam consists of two lobes of comparable size oriented frontally and ventrally. In level flight, the bat directs its beam such that the ventral lobe is pointed forward and down toward the ground ahead of its flight path. The bat may utilize the downward directed lobe to measure altitude without the need for vertical head movements.

Echolocating bats use a nearly time-optimal strategy to intercept prey.

Acquisition of food in many animal species depends on the pursuit and capture of moving prey. Among modern humans, the pursuit and interception of moving targets plays a central role in a variety of sports, such as tennis, football, Frisbee, and baseball. Studies of target pursuit in animals, ranging from dragonflies to fish and dogs to humans, have suggested that they all use a constant bearing (CB) strategy to pursue prey or other moving targets. CB is best known as the interception strategy employed by baseball outfielders to catch ballistic fly balls. CB is a time-optimal solution to catch targets moving along a straight line, or in a predictable fashion--such as a ballistic baseball, or a piece of food sinking in water. Many animals, however, have to capture prey that may make evasive and unpredictable maneuvers. Is CB an optimum solution to pursuing erratically moving targets? Do animals faced with such erratic prey also use CB? In this paper, we address these questions by studying prey capture in an insectivorous echolocating bat. Echolocating bats rely on sonar to pursue and capture flying insects. The bat's prey may emerge from foliage for a brief time, fly in erratic three-dimensional paths before returning to cover. Bats typically take less than one second to detect, localize and capture such insects. We used high speed stereo infra-red videography to study the three dimensional flight paths of the big brown bat, Eptesicus fuscus, as it chased erratically moving insects in a dark laboratory flight room. We quantified the bat's complex pursuit trajectories using a simple delay differential equation. Our analysis of the pursuit trajectories suggests that bats use a constant absolute target direction strategy during pursuit. We show mathematically that, unlike CB, this approach minimizes the time it takes for a pursuer to intercept an unpredictably moving target. Interestingly, the bat's behavior is similar to the interception strategy implemented in some guided missiles. We suggest that the time-optimal strategy adopted by the bat is in response to the evolutionary pressures of having to capture erratic and fast moving insects.

Steering by hearing: a bat's acoustic gaze is linked to its flight motor output by a delayed, adaptive linear law.

Adaptive behaviors require sensorimotor computations that convert information represented initially in sensory coordinates to commands for action in motor coordinates. Fundamental to these computations is the relationship between the region of the environment sensed by the animal (gaze) and the animal's locomotor plan. Studies of visually guided animals have revealed an anticipatory relationship between gaze direction and the locomotor plan during target-directed locomotion. Here, we study an acoustically guided animal, an echolocating bat, and relate acoustic gaze (direction of the sonar beam) to flight planning as the bat searches for and intercepts insect prey. We show differences in the relationship between gaze and locomotion as the bat progresses through different phases of insect pursuit. We define acoustic gaze angle, theta(gaze), to be the angle between the sonar beam axis and the bat's flight path. We show that there is a strong linear linkage between acoustic gaze angle at time t [theta(gaze)(t)] and flight turn rate at time t + tau into the future [theta(flight) (t + tau)], which can be expressed by the formula theta(flight) (t + tau) = ktheta(gaze)(t). The gain, k, of this linkage depends on the bat's behavioral state, which is indexed by its sonar pulse rate. For high pulse rates, associated with insect attacking behavior, k is twice as high compared with low pulse rates, associated with searching behavior. We suggest that this adjustable linkage between acoustic gaze and motor output in a flying echolocating bat simplifies the transformation of auditory information to flight motor commands.

The sonar beam pattern of a flying bat as it tracks tethered insects.

This paper describes measurements of the sonar beam pattern of flying echolocating bats, Eptesicus fuscus, performing various insect capture tasks in a large laboratory flight room. The beam pattern is deduced using the signal intensity across a linear array of microphones. The positions of the bat and insect prey are obtained by stereoscopic reconstruction from two camera views. Results are reported in the form of beam-pattern plots and estimated direction of the beam axis. The bat centers its beam axis on the selected target with a standard deviation (sigma) of 3 degrees. The experimental error is +/- 1.4 degrees. Trials conducted with two targets show that the bat consistently tracks one of the targets with its beam. These findings suggest that the axis of the bat sonar beam is a good index of selective tracking of targets, and in this respect is analogous to gaze in predominantly visual animals.

Research, robots, and reality: A statement on current trends in biorobotics.

While robotics has benefited from inspiration gained from biology, the opposite is not the case: there are few if any cases in which robotic models have lead to genuine insight into biology. We analyze the reasons why biorobotics has been essentially a one-way street. We argue that the development of better tools is essential for progress in this field.