Czechia

Genome specific association study (GSAS) for exploration of intravarietal variability in hemp (Cannabis sativa)

Trubanová Nina (1); Isobe Sachiko (2); Shirasawa, Kenta (2); Watanabe, Akiko (2); Melzer, Rainer (1, 3), Schilling, Susanne (1, 3)

1 School of Biology and Environmental Science, University College Dublin, Science Centre West, Belfield, Dublin 4, Republic of Ireland; 2 Laboratory of Plant Genetics and Genomics, Department of Frontier Research and Development, Kazusa DNA Research Institute; 3 UCD Earth Institute, University College Dublin

Hemp (Cannabis sativa L.) stands out as a versatile crop with substantial economic potential. However, its intravarietal phenotypic and genetic variability remain inadequately elucidated. In the context of advancing crop improvement efforts, our research addresses this gap by employing an innovative genome-specific association study (GSAS) framework.
The GSAS methodology involves the streamlined generation of a single phenotyped population, the selection of alleles heterozygous in the parent plant, and the application of a model conducive to the polygenic association of studied traits. Through extensive exploration of plant growth, development, and reproductive patterns, along with genetic variability analysis of a population derived from a single plant, we identified statistically significant single nucleotide variants and haplotypes associated with studied traits.
Here presented study highlights the potential of the GSAS approach for utilising genomics and phenomics to significantly advance our understanding of the genetic determination of intravarietal phenotypic variability in hemp and other highly heterozygous crops. The findings from prospective GSAS studies hold promise for marker-assisted breeding, enabling the development of new cultivars with enhanced uniformity and improved performance in traits relevant to various applications, ranging from pharmaceuticals to the manufacturing of materials with superb properties, adaptable to various climates.

SEB Conference Prague 2024

Genome specific association study (GSAS) for exploration of intravarietal variability in hemp (Cannabis sativa)

traits

genotype

gsas

hemp

cannabis sativa

phenotype

variability

technical paper

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Ammonia is a toxic nitrogenous waste causing ionoregulatory and neuronal dysfunction in aquatic animals. Ammonotelic animals excrete ammonia as is and must have therefore an efficient excretion process in place to keep blood/hemolymph ammonia levels in a tolerable range. While in fish and crustaceans, the excretory mechanism has been investigated in greater detail, the cellular regulation of these processes is rather unknown. To investigate which cellular pathways are involved in ammonia excretion, gills from osmoregulating green crabs were perfused with a hemolymph-like solution containing 500µM NH4Cl, mimicking hemolymph levels found after feeding. Activation of the PKA pathway by adding either Forskolin or 8-Bromo-cAMP, which is known to activate osmoregulatory NaCl uptake in crustaceans, to the perfusion solution caused a significant decrease in the ammonia excretion rate. By contrast, activating the PKG pathway by the addition of 8-Bromo-cGMP, caused an increase of excretion, which was blocked when PKG activity was inhibited via KT5823. When no ammonia was added to either side of the branchial epithelium, metabolic ammonia was released predominantly to the environment. Interestingly, the addition of 8-Bromo-cAMP caused not only an increase of ammoniagenesis, it also promoted that a higher proportion of metabolic ammonia is released towards the hemolymph side, while apical release remained unaltered.

The cGMP/PKG pathway, but not the cAMP/PKA pathway promotes branchial ammonia excretion in the green shore crab

The respiratory and metabolic systems of insects have been shown to exhibit developmental plasticity when exposed to alterations in oxygen availability during development. For example, adult trachea and mitochondria density in Drosophila is dependent on ambient oxygen levels experienced during larval development. Manduca sexta are large, endothermic moths that require elevated thorax temperatures for flight. Here we asked how energy demands of this endothermic insect influenced the developmental plasticity of the metabolic system under either hypoxic or hyperoxic developmental conditions. Manduca sexta were reared in either 10, 21, or 30% oxygen from the larval stage through adults. We examined the plasticity of tracheal and mitochondrial morphology and physiology of the adult moth. Hypoxic reared moths reached smaller sizes as adults while hyperoxic reared moths reached larger adult sizes compared with normoxic reared animals. Both hypoxic and hyperoxic reared moths had significantly longer pupation times than controls. Hypoxic reared moths had lower critical oxygen levels for flight compared with hyperoxic reared moths. Mitochondrial respiration rate in permeabilized flight muscle mitochondria was higher in hypoxic reared moths than in either control or hyperoxic reared moths, but this difference was not observed in isolated mitochondria. Unlike Drosophila, Manduca exhibit no developmental plasticity in mitochondrial densities or tracheal densities in response to hyperoxia and hypoxia. The developing Manduca sexta exhibits more limited plasticity in resopnse to altered oxygen when compared with other insects studied.

Are there constraints on the developmental plasticity of metabolic physiology in an endothermic insect?

Climate change is one of the greatest challenges of our era, and identifying the most vulnerable populations is urgently needed to guide conservation efforts. Although over 40% of amphibian species are currently listed as threatened, our understanding of their resilience to rising temperatures remains limited. This is primarily because knowledge of thermal tolerance is taxonomically and geographically biased. Data on amphibian heat tolerance limits only covers 7.5% of known species and is geographically biased towards temperate regions. This discrepancy is problematic, considering the high species richness in the tropics and the mounting evidence that tropical ectotherms are most susceptible to rising temperatures. In this study, we aimed to resolve existing biases in physiological data to assess the vulnerability of amphibians to global warming at truly global scales. We first employed a novel data imputation approach to predict the heat tolerance of 60% of amphibian species with high accuracy. We then assessed their vulnerability to daily temperature variation in different thermal refugia (terrestrial, aquatic, arboreal) using biophysical models. We found that 198 out of 5203 species are already exposed to overheating events in shaded terrestrial conditions. Despite accounting for heat tolerance plasticity, a 4°C global temperature increase would create a step-change in impact severity, pushing 9.4% of species beyond their physiological limits. In the Southern Hemisphere, tropical species encounter disproportionally more overheating events, while in the Northern Hemisphere, non-tropical species are more susceptible. Our findings challenge common evidence for latitudinal gradients in overheating risk and underscore the importance of considering climatic variability in vulnerability assessments. Importantly, our predictions lead to clear directives for research and management - pointing to specific populations and species with increased risk, predominantly in under-studied regions of the Global South. Our microclimate-explicit analyses also demonstrate the crucial role vegetation and water bodies play in mitigating the impacts of heat waves on amphibians. Immediate action is needed to preserve and manage these vital microhabitat features.

How vulnerable are amphibians to global warming? 

The skin microbiome is an integral part of its host, capable of aiding in wound healing and defending against pathogens. While the skin microbiome is susceptible to changes due to contact with the environment, it has also been shown to be host- or species-specific, implying that certain microbes play defined roles in the host-microbiome relationship. Bats present an interesting study system for exploring both the protective role and temporal changes in the skin microbiome, as bats alternate between active periods and hibernation throughout the year. During hibernation, a bat’s immune system becomes downregulated, making it more vulnerable to infection by surrounding pathogens. Despite constant exposure to the fungal pathogen responsible for white-nose syndrome in Nearctic bats, Palearctic bats remain uninfected. Symbionts residing on the skin of Palearctic bats may help mitigate infection by increasing the abundance of protective antifungal bacteria, thus defending the bats from fatal infection. To investigate whether the skin microbiome of bats could potentially become more protective during hibernation, we collected swab samples from five European bat species during both the active period and hibernation. A comparison of the sequenced samples will reveal whether the bacterial composition on the skin shifts to contain more bacteria with described antifungal properties during winter, when bats are torpid and exposed to the white-nose pathogen. This information will advance our understanding of how Palearctic bats survive exposure to the deadly fungus and could potentially be used in the development of probiotic treatments aimed at mitigating white-nose syndrome in Nearctic bats.

Seasonal shifts in the skin microbiome of bats and their relevance to bat immunity during hibernation

Experimental variation is a multi-faceted phenomenon leading to both intra-experimental and inter-experimental incongruencies. Such variation often manifests in nonreproducibility of experiments which is rightfully regarded as an issue in experimental sciences. Nonreproducibility has been attributed to inadequate reporting of methods, sloppiness/honest error, and even misconduct. We hypothesized that, within the field of animal physiology, the most parsimonious explanation for nonreproducibility is inadequate reporting of key methodological details. We analyzed 84 research articles published in five primarily organismal animal physiology journals and assessed compliance for 34 reporting variables referring to biology, experimentation, and data collection/analysis. Our study shows that about one third of necessary information on the methodology is missing. We thus assume that at least within the life sciences, underreporting of key variables can lead to a lack of reproducibility that is independent of the rigor of the study, but rather reflects the lack of rigor of the journal article preparation as well as peer review and publishing.

Addressing Nonreproducibility: Examining Methodological Reporting in Animal Physiology Studies

Regulating the Organelle Band Organization in Male Meiosis of Arabidopsis
Yingrui Ma
Meiosis is a unique cell division process for sexual reproduction in plants and animals. In the meiotic cell cycle, a single round of DNA replication is followed by two rounds of chromosome division, called meiosis I and meiosis II. After Meiosis I, the divided homologous chromosomes are separated by the newly synthesized cell wall or a cell wall like structure. In dicotyledons, instead of a real cell wall, a band structure consisting of vesicles derived from different organelles, called an organelle band, is formed after the first chromosome division. The disruption of this structure in meiosis II can result in nuclear restitution and production of unreduced gametes. The mutation that leads to the disorder of the organelle band is named jason (jas). In this study, a suppressor of jas, called pel, was obtained through forward genetic screening. PEL is a vesicle membrane located protein, and located in the cytoplasm during meiotic cell cycle. The recovered organelle band in jas pel is sensitive to exocytotic vesicle trafficking inhibitors, suggesting that exocytotic vesicle trafficking pathway is important for organelle band maintenance in jas pel. Furthermore, destabilized actin filaments are found to partially recover the jas phenotype. vesicles can be transferred through actin filament, PEL and PEL interaction proteins transfer of exocytotic vesicles through actin filaments within male meiocytes. Taken together, this research suggests a process which involved exocytotic vesicle trafficking and actin dynamic to maintain the organelle band, acting as a physical barrier to prevent nuclear restitution during male meiosis.

Regulating the Organelle Band Organization in Male Meiosis of Arabidopsis

Plants are sessile organisms adapting constantly to environmental fluctuations through spatial and temporal transcriptional responses. We used the latest generation of MS2 RNA labelling system combined with microfluidics to monitor impact of modification of phosphate supply by live transcription imaging. MS2/MCT system is based on a fusion between GFP and the coat protein of bacteriophage MS2 (MCP-GFP), which recognizes a specific RNA stem-loop inserted in multiple copies into a reporter RNA. This enabled quantitative measurements of the transcriptional activity of single loci in living Arabidopsis plants, providing direct visualization of transcriptional regulation within entire organs. Using phosphate responsive genes as model, we found that active genes displayed high transcription initiation rates and frequently clustered together in endo-replicated cells. We also observed large differences between alleles of single cells and in agreement, intrinsic noise was larger than extrinsic variations. Moreover, we established that the transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpected fast kinetics in the range of minutes, and with striking heterogeneity between neighboring cells. Access to single cells RNA polymerase II dynamics within live plants will benefit the study of numerous signaling processes.

RNA live imaging: interest for plant biology

Temperature is a key determinant of plant distribution and phenology. Warmer temperatures due to climate change have already altered the flowering times of wild plants, and crop yields decline significantly with additional temperature stress. Despite the importance of temperature on plant growth and development, little is understood of the mechanisms of temperature sensing in plants. We have identified thermosensory nodes that can account for key warm temperature responses for both stress and developmental pathways. We have found that protein phase change appears to play a role in several temperature response pathways, and I will describe some of these in Arabidopsis thaliana.

Temperature sensing in plants

Plants experience heat stress (HS) at temperatures where growth and development are negatively affected. Survival from HS is dependent on the upregulation of hundreds of genes with protective functions for protein and cellular homeostasis such as heat shock proteins (HSPs). The majority of these genes are regulated by the family of HS transcription factors (HSF). In tomato, HsfA1a is the master regulator of HS response. Additional HSFs, such as HsfA2 and HsfA7 are important for HS acclimation and memory, the ability of a plant to store the information of a past HS incident and optimize the response in case of a new one. Class B HSFs act as co-activators and co-repressors and regulate the intensity and the timely attenuation of the stress response to optimize recovery. In addition to transcription, RNA splicing is another important mechanism for regulation of gene expression under HS conditions. Our results suggest that thermotolerance requires the coordinated but also independent activity of transcription factors and splicing regulators to optimize the cellular reprogramming required for survival under high temperatures.

Heat stress transcription factors and splicing regulators: brothers in arms to survive heat

This talk is split into two parts. I will first examine whether there is sufficient evidence for male ejaculate senescence across the animal kingdom. I will present data from a meta-analysis we conducted on 379 studies, to quantify the effects of advancing male age on ejaculate traits across 157 species of non-human animals. This work shows that there is high heterogeneity in the available data, hence I will try to tease apart the factors that might explain this variation. In the second part of the talk, I will focus on ejaculate and reproductive senescence in the model organism Drosophila melanogaster and try to identify the molecular drivers, contributing factors and fitness consequences of advancing male age for the male, its female mate and for its offspring.

Genome specific association study (GSAS) for exploration of intravarietal variability in hemp (Cannabis sativa)

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The cGMP/PKG pathway, but not the cAMP/PKA pathway promotes branchial ammonia excretion in the green shore crab

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Genome specific association study (GSAS) for exploration of intravarietal variability in hemp (Cannabis sativa)

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Next from SEB Conference Prague 2024

The cGMP/PKG pathway, but not the cAMP/PKA pathway promotes branchial ammonia excretion in the green shore crab

Stay up to date with the latest Underline news!

PRESENTATIONS

CONFERENCES

COMPANY

RESOURCES

Downloads