ITErRoot: High Throughput Segmentation of 2-Dimensional Root System Architecture

Root system architecture (RSA) analysis is a form of high-throughput plant phenotyping which has recently benefited from the application of various deep learning techniques. A typical RSA pipeline includes a segmentation step, where the root system is extracted from 2D images. The segmented image is then passed to subsequent steps for processing, which result in some representation of the architectural properties of the root system. This representation is then used for trait computation, which can be used to identify various desirable properties of a plant’s RSA. Errors which arise at the segmentation stage can propagate themselves throughout the remainder of the pipeline and impact results of trait analysis. This work aims to design an iterative neural network architecture, called ITErRoot, which is particularly well suited to the segmentation of root structure from 2D images in the presence of non-root objects. A novel 2D root image dataset is created along with a ground truth annotation tool designed to facilitate consistent manual annotation of RSA. The proposed architecture is able to take advantage of the root structure to obtain a high quality segmentation and is generalizable to root systems with thin roots, showing improved quality over recent approaches to RSA segmentation. We provide rigorous analysis designed to identify the strengths and weaknesses of the proposed model as well as to validate the effectiveness of the approach for producing high-quality segmentations

Geometric Algorithms for Modeling Plant Roots from Images

Roots, considered as the ”hidden half of the plant”, are essential to a plant’s health and pro- ductivity. Understanding root architecture has the potential to enhance efforts towards im- proving crop yield. In this dissertation we develop geometric approaches to non-destructively characterize the full architecture of the root system from 3D imaging while making com- putational advances in topological optimization. First, we develop a global optimization algorithm to remove topological noise, with applications in both root imaging and com- puter graphics. Second, we use our topology simplification algorithm, other methods from computer graphics, and customized algorithms to develop a high-throughput pipeline for computing hierarchy and fine-grained architectural traits from 3D imaging of maize roots. Finally, we develop an algorithm for consistently simplifying the topology of nested shapes, with a motivating application in temporal root system analysis. Along the way, we con- tribute to the computer graphics community a pair of topological simplification algorithms both for repairing a single 3D shape and for repairing a sequence of nested shapes

Plant Responses to Stress and Environmental Stimulus

Plants respond to diverse environmental stimuli such as light, nutrients, temperature, and oxygen, which shape their growth and fate. When these stimuli are suboptimal for adequate plant growth, they cause stress. This book is a collection of research articles providing evidence about plant responses to stresses and environmental stimuli, as well as new methodologies for plant phenotyping

Soil composition and rootstock genotype drive the root associated microbial communities in young grapevines

Soil microbiota plays a significant role in plant development and health and appears to be a major component of certain forms of grapevine decline. A greenhouse experiment was conducted to study the impact of the microbiological quality of the soil and grapevine rootstock genotype on the root microbial community and development of young plants. Two rootstocks heterografted with the same scion were grown in two vineyard soils differing in microbial composition and activities. After 4 months, culture-dependent approaches and amplicon sequencing of bacterial 16S rRNA gene and fungal ITS were performed on roots, rhizosphere and bulk soil samples. The root mycorrhizal colonization and number of cultivable microorganisms in the rhizosphere compartment of both genotypes were clearly influenced by the soil status. The fungal diversity and richness were dependent on the soil status and the rootstock, whereas bacterial richness was affected by the genotype only. Fungal genera associated with grapevine diseases were more abundant in declining soil and related root samples. The rootstock affected the compartmentalization of microbial communities, underscoring its influence on microorganism selection. Fluorescence in situ hybridization (FISH) confirmed the presence of predominant root-associated bacteria. These results emphasized the importance of rootstock genotype and soil composition in shaping the microbiome of young vines

Discovering Novel Polyextremotolerant Fungi, and Determining their Ecological Role Within the Biological Soil Crust Consortium

The ecological niche of polyextremotolerant fungi within oligotrophic ecosystems such as biological soil crusts has not yet been determined. These fungi persist in locations where nutrients are depleted while simultaneously surrounded by autotrophic microbes such as algae and cyanobacteria. Yet it has not been shown that they are engaging in any exchange of nutrients the way lichens do. However, there is seemingly no other way for these fungi to obtain vital nutrients, such as carbon or nitrogen, other than from these microbes. Here we have isolated polyextremotolerant fungi from cold desert biological soil crusts which are a microbial biofilm that form on the surfaces of non-vegetative soils and contain an abundance of autotrophic microbes. The presence of free-living fungi in these biofilms has recently been verified, but only a few fungi have been cultured directly from them, therefore the ecological role of fungi in the biological soil crust remains unknown. With work presented here, we have shown that polyextremotolerant fungi are present within the biological soil crust. Additionally, we have provided potential leads to the ecological niches of these organisms within the biological soil crust. Exophiala viscosium and Exophiala limosus, tentatively named, are two novel species described here, which have been observed to secrete excess amounts of melanin into their media. Since melanin is a carbon-expensive product to make, we believe they are secreting it to protect the biological soil crust from UV and desiccation. Additionally, we have identified what we believe to be a polyextremotolerant fungus with endosymbiotic bacteria, Crusty and its Methylobacterium symbionts Light Pinky and Dark Pinky. While we have not directly confirmed the basis of their endosymbiosis, we believe it is to allow the bacteria to optimally perform aerobic anoxygenic photosynthesis and auxin production, due to the genetic confirmation of the mechanisms required for these processes within the genomes of these bacteria, and the significant increase in active metabolism of Crusty-Pinky when grown in the presence of light. Detailed descriptions of the methods of experiments performed and the results of this study will provide a basis for research in the future on polyextremotolerant fungi, and determine the microbial interactions that allow them to survive oligotrophic conditions. Advisors: Wayne R. Riekhof & Steven D. Harri

Phylogenetics in the Genomic Era

Molecular phylogenetics was born in the middle of the 20th century, when the advent of protein and DNA sequencing offered a novel way to study the evolutionary relationships between living organisms. The first 50 years of the discipline can be seen as a long quest for resolving power. The goal – reconstructing the tree of life – seemed to be unreachable, the methods were heavily debated, and the data limiting. Maybe for these reasons, even the relevance of the whole approach was repeatedly questioned, as part of the so-called molecules versus morphology debate. Controversies often crystalized around long-standing conundrums, such as the origin of land plants, the diversification of placental mammals, or the prokaryote/eukaryote divide. Some of these questions were resolved as gene and species samples increased in size. Over the years, molecular phylogenetics has gradually evolved from a brilliant, revolutionary idea to a mature research field centred on the problem of reliably building trees. This logical progression was abruptly interrupted in the late 2000s. High-throughput sequencing arose and the field suddenly moved into something entirely different. Access to genome-scale data profoundly reshaped the methodological challenges, while opening an amazing range of new application perspectives. Phylogenetics left the realm of systematics to occupy a central place in one of the most exciting research fields of this century – genomics. This is what this book is about: how we do trees, and what we do with trees, in the current phylogenomic era. One obvious, practical consequence of the transition to genome-scale data is that the most widely used tree-building methods, which are based on probabilistic models of sequence evolution, require intensive algorithmic optimization to be applicable to current datasets. This problem is considered in Part 1 of the book, which includes a general introduction to Markov models (Chapter 1.1) and a detailed description of how to optimally design and implement Maximum Likelihood (Chapter 1.2) and Bayesian (Chapter 1.4) phylogenetic inference methods. The importance of the computational aspects of modern phylogenomics is such that efficient software development is a major activity of numerous research groups in the field. We acknowledge this and have included seven "How to" chapters presenting recent updates of major phylogenomic tools – RAxML (Chapter 1.3), PhyloBayes (Chapter 1.5), MACSE (Chapter 2.3), Bgee (Chapter 4.3), RevBayes (Chapter 5.2), Beagle (Chapter 5.4), and BPP (Chapter 5.6). Genome-scale data sets are so large that statistical power, which had been the main limiting factor of phylogenetic inference during previous decades, is no longer a major issue. Massive data sets instead tend to amplify the signal they deliver – be it biological or artefactual – so that bias and inconsistency, instead of sampling variance, are the main problems with phylogenetic inference in the genomic era. Part 2 covers the issues of data quality and model adequacy in phylogenomics. Chapter 2.1 provides an overview of current practice and makes recommendations on how to avoid the more common biases. Two chapters review the challenges and limitations of two key steps of phylogenomic analysis pipelines, sequence alignment (Chapter 2.2) and orthology prediction (Chapter 2.4), which largely determine the reliability of downstream inferences. The performance of tree building methods is also the subject of Chapter 2.5, in which a new approach is introduced to assess the quality of gene trees based on their ability to correctly predict ancestral gene order. Analyses of multiple genes typically recover multiple, distinct trees. Maybe the biggest conceptual advance induced by the phylogenetic to phylogenomic transition is the suggestion that one should not simply aim to reconstruct “the” species tree, but rather to be prepared to make sense of forests of gene trees. Chapter 3.1 reviews the numerous reasons why gene trees can differ from each other and from the species tree, and what the implications are for phylogenetic inference. Chapter 3.2 focuses on gene trees/species trees reconciliation methods that account for gene duplication/loss and horizontal gene transfer among lineages. Incomplete lineage sorting is another major source of phylogenetic incongruence among loci, which recently gained attention and is covered by Chapter 3.3. Chapter 3.4 concludes this part by taking a user’s perspective and examining the pros and cons of concatenation versus separate analysis of gene sequence alignments. Modern genomics is comparative and phylogenetic methods are key to a wide range of questions and analyses relevant to the study of molecular evolution. This is covered by Part 4. We argue that genome annotation, either structural or functional, can only be properly achieved in a phylogenetic context. Chapters 4.1 and 4.2 review the power of these approaches and their connections with the study of gene function. Molecular substitution rates play a key role in our understanding of the prevalence of nearly neutral versus adaptive molecular evolution, and the influence of species traits on genome dynamics (Chapter 4.4). The analysis of substitution rates, and particularly the detection of positive selection, requires sophisticated methods and models of coding sequence evolution (Chapter 4.5). Phylogenomics also offers a unique opportunity to explore evolutionary convergence at a molecular level, thus addressing the long-standing question of predictability versus contingency in evolution (Chapter 4.6). The development of phylogenomics, as reviewed in Parts 1 through 4, has resulted in a powerful conceptual and methodological corpus, which is often reused for addressing problems of interest to biologists from other fields. Part 5 illustrates this application potential via three selected examples. Chapter 5.1 addresses the link between phylogenomics and palaeontology; i.e., how to optimally combine molecular and fossil data for estimating divergence times. Chapter 5.3 emphasizes the importance of the phylogenomic approach in virology and its potential to trace the origin and spread of infectious diseases in space and time. Finally, Chapter 5.5 recalls why phylogenomic methods and the multi-species coalescent model are key in addressing the problem of species delimitation – one of the major goals of taxonomy. It is hard to predict where phylogenomics as a discipline will stand in even 10 years. Maybe a novel technological revolution will bring it to yet another level? We strongly believe, however, that tree thinking will remain pivotal in the treatment and interpretation of the deluge of genomic data to come. Perhaps a prefiguration of the future of our field is provided by the daily monitoring of the current Covid-19 outbreak via the phylogenetic analysis of coronavirus genomic data in quasi real time – a topic of major societal importance, contemporary to the publication of this book, in which phylogenomics is instrumental in helping to fight disease