Browsing by Author "Haak, David C."

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    Adaptation of an invasive grass to agriculture: ecological and genomic evidence
    Smith, Alyssa Laney (Virginia Tech, 2017-06-06)
    Species vary phenotypically and genetically across their environmental range limits, and this variation can influence ecological processes. Ecologically meaningful intraspecific variation might be particularly important in the context of agricultural weeds and exotic invaders, because intraspecific variation in these species might allow them to rapidly adapt to their unusually dynamic and variable environments. In a greenhouse study, we explored intraspecific variation in the size, rhizome production, and competitive ability of the global invader, Johnsongrass (Sorghum halepense), representing populations from agricultural and non-agricultural habitats across its introduced North American range. We also used these populations to explore the relationship between phenotypic variation and genomic endoreduplication responses to the common stresses herbicides, competition, and clipping. Endoreduplication occurs when plants increase their genome size by increasing their nuclear chromosome number, with some evidence showing correlations with stress response. We found that Johnsongrass plants from agricultural habitats were larger than plants from non-agricultural habitats, but there was no difference between habitats in either rhizome production or competitive ability. Two of the five herbicides we tested, primisulfuron and imazethapyr, had the strongest suppressive effects on Johnsongrass, and also stimulated the greatest rates of endoreduplication. Furthermore, agricultural populations showed higher levels of endoreduplication. We found no overall effect of competition on endoreduplication, although endoreduplication was higher for non-agricultural populations than agricultural populations. When competing with corn, but not with conspecifics, Johnsongrass roots increased endoreduplication by 13%. Clipping induced substantial endoreduplication, but there was no difference between agricultural and non-agricultural populations. Our results suggest that endoreduplication may play a role in some, but not all, stress responses in Johnsongrass. Furthermore, our results indicate that Johnsongrass has adapted in some phenotypic and genomic ways to agricultural habitats in North America. Such adaptation may play a role in this species' success as both an agricultural weed and an exotic invader.
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    Adaptive constraints at the range edge of a widespread and expanding invasive plant
    Fletcher, Rebecca A.; Atwater, Daniel Z.; Haak, David C.; Bagavathiannan, Muthukumar; DiTommaso, Antonio; Lehnhoff, Erik; Paterson, Andrew H.; Auckland, Susan; Govindasamy, Prabhu; Lemke, Cornelia; Morris, Edward; Rainville, Lisa; Barney, Jacob N. (Oxford University Press, 2023-11-05)
    Identifying the factors that facilitate and limit invasive species' range expansion has both practical and theoretical importance, especially at the range edges. Here, we used reciprocal common garden experiments spanning the North/South and East/West range that include the North American core, intermediate and range edges of the globally invasive plant, Johnsongrass (Sorghum halepense) to investigate the interplay of climate, biotic interactions (i.e. competition) and patterns of adaptation. Our results suggest that the rapid range expansion of Johnsongrass into diverse environments across wide geographies occurred largely without local adaptation, but that further range expansion may be restricted by a fitness trade-off that limits population growth at the range edge. Interestingly, plant competition strongly dampened Johnsongrass growth but did not change the rank order performance of populations within a garden, though this varied among gardens (climates). Our findings highlight the importance of including the range edge when studying the range dynamics of invasive species, especially as we try to understand how invasive species will respond to accelerating global changes.
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    Adaptive evolution, sex-linkage, and gene conversion in the voltage-gated sodium channels of toxic newts and their snake predators
    Gendreau, Kerry (Virginia Tech, 2022-05-27)
    Understanding how genetic changes ultimately affect morphology and physiology is essential for understanding and predicting how organisms will adapt to environmental changes. Although most traits are complex and involve the interplay of many different genetic loci, some exceptions exist. These include the convergent evolution of tetrodotoxin resistance in snakes, which has a simple genetic basis and can be used as a model system to investigate the genetic basis of adaptive evolution. Tetrodotoxin is a potent neurotoxin used as a chemical defense by various animals, including toxic newts. Snakes have evolved resistance through mutations in voltage-gated sodium channels, the protein targets of tetrodotoxin, sparking an evolutionary arms race between predator and prey. In this dissertation, I describe how genomic rearrangements have led to sex-linkage of four of the voltage-gated sodium channel genes in snakes and compare allele frequencies across populations and sexes to make inferences about how sex linkage has influenced the evolution of resistance in garter snakes. By measuring gene expression in different snake tissues, I show that three of these sex-linked sodium channel genes are dosage compensated in embryos, adult muscle, and adult brain. In contrast, two channels show sexual dimorphism in their expression levels in the heart, which may indicate differences in dosage compensation among tissues. I then use comparative genomics to track the evolutionary history of tetrodotoxin resistance across all nine sodium channel genes in squamate reptiles and show how historical changes have paved the way for full-body resistance in certain snakes. Finally, I use targeted sequence capture to obtain the sodium channel sequences of salamanders and show evidence that tetrodotoxin self-resistance in toxic newts was likely accelerated through gene conversion between resistant and non-resistant sodium channel paralogs. Together, these results illustrate parallelism in evolutionary mechanisms and processes contributing to the appearance of an extreme and complex trait that arose independently in two distinct taxa separated by hundreds of millions of years.
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    Aggregation Pheromone Biosynthesis and Engineering in Plants for Stinkbug Pest Management
    Lehner, Bryan W. (Virginia Tech, 2019-04-26)
    Stinkbugs (Pentatomidae) and other agricultural pests such as bark beetles and flea beetles are known to synthesize terpenoids as aggregation pheromones. Knowledge of the genes and enzymes involved in pheromone biosynthesis may allow engineering of pheromone biosynthetic pathways in plants to develop new forms of trap crops and agricultural practices for pest management. The harlequin bug, Murgantia histrionica, a specialist pest on crucifer crops, produces the sesquiterpene, murgantiol, as a male-specific aggregation pheromone. Similarly, the southern green stink bug, Nezara viridula, a generalist pest worldwide on soybean and other crops, releases sesquiterpene cis-/trans-(Z)-α-bisabolene epoxides as male-specific aggregation pheromone. In both species, enzymes called terpene synthases (TPSs) synthesize precursors of the aggregation pheromones, which are sesquipiperitol and (Z)-α-bisabolene as the precursor of murgantiol and cis-/trans-(Z)-α-bisabolene epoxide, respectively. We hypothesized that enzymes in the family of cytochrome P450 monooxygenases are involved in the conversion of these precursors to the final epoxide products. This study investigated the tissue specificity and sequence of these conversions by performing crude enzyme assays with protein extracts from male tissues. Furthermore, candidate P450 genes were selected by RNA-sequencing and co-expression analysis and the corresponding recombinant proteins tested for enzyme activity. To engineer the pheromone biosynthetic enzymes in plants, transient expression of the TPSs of both stink bugs was performed in Nicotiana benthamiana leaves. Both sesquipiperitol and (Z)-α-bisabolene were found to be produced and emitted from inoculated N. benthamiana leaves. Future work will implement stable transformation to engineer murgantiol biosynthesis in crucifer trap crops and develop similar approaches for pheromone engineering of other agricultural pests.
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    Assessing age, breeding stage, and mating activity as drivers of variation in the reproductive microbiome of female tree swallows
    Hernandez, Jessica; Hucul, Catherine; Reasor, Emily; Smith, Taryn; McGlothlin, Joel W.; Haak, David C.; Belden, Lisa K.; Moore, Ignacio T. (Wiley, 2021-07)
    Sexually transmitted microbes are hypothesized to influence the evolution of reproductive strategies. Though frequently discussed in this context, our understanding of the reproductive microbiome is quite nascent. Indeed, testing this hypothesis first re-quires establishing a baseline understanding of the temporal dynamics of the reproductive microbiome and of how individual variation in reproductive behavior and age influence the assembly and maintenance of the reproductive microbiome as a whole. Here, we ask how mating activity, breeding stage, and age influence the reproductive microbiome. We use observational and experimental approaches to explain variation in the cloacal microbiome of free- living, female tree swallows (Tachycineta bicolor). Using microsatellite- based parentage analyses, we determined the number of sires per brood (a proxy for female mating activity). We experimentally increased female sexual activity by administering exogenous 17ß-estradiol. Lastly, we used bacterial 16S rRNA amplicon sequencing to characterize the cloacal microbiome. Neither the number of sires per brood nor the increased sexual activity of females significantly influenced female cloacal microbiome richness or community structure. Female age, however, was positively correlated with cloacal microbiome richness and influenced overall community structure. A hypothesis to explain these patterns is that the effect of sexual activity and the number of mates on variation in the cloacal microbiome manifests over an individual's lifetime. Additionally, we found that cloacal microbiome alpha diversity (Shannon Index, Faith's phylogenetic distance) decreased and community structure shifted between breeding stages. This is one of few studies to document within-individual changes and age- related differences in the cloacal microbiome across successive breeding stages. More broadly, our results contribute to our understanding of the role that host life history and behavior play in shaping the cloacal microbiomes of wild birds.
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    Assessment of Genetic Diversity and Population Structure in Iranian Cannabis Germplasm
    Soorni, Aboozar; Fatahi, Reza; Haak, David C.; Salami, Seyed Alireza; Bombarely, Aureliano (Nature Publishing Group, 2017-11-15)
    Cannabis sativa has a complex history reflected in both selection on naturally occurring compounds and historical trade routes among humans. Iran is a rich resource of natural populationswhich hold the promise to characterize historical patterns of population structure and genetic diversity within Cannabis. Recent advances in high-throughput DNA sequencing technologies have dramatically increased our ability to produce information to the point that it is now feasible to inexpensively obtain population level genotype information at a large scale. In the present investigation, we have explored the use of Genotyping-By-Sequencing (GBS) in Iranian cannabis. We genotyped 98 cannabis samples 36 from Iranian locations and 26 accessions from two germplasm collections. In total, 24,710 high-quality Single Nucleotide Polymorphisms (SNP) were identified. Clustering analysis by Principal Component Analysis (PCA) identified two genetic clusters among Iranian populations and fineSTRUCTURE analysis identified 19 populations with some geographic partitioning. We defined Iranian cannabis in two main groups using the results of the PCA and discovered some strong signal to define some locations as population according to fineSTRUCTURE analyses. However, single nucleotide variant analysis uncovered a relatively moderate level of variation among Iranian cannabis.
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    Bacteriophages in the honey bee gut and amphibian skin microbiomes: investigating the interactions between phages and their bacterial hosts
    Bueren, Emma Kathryn Rose (Virginia Tech, 2024-06-14)
    The bacteria in host-associated microbial communities influence host health through various mechanisms, such as immune stimulation or the release of metabolites. However, viruses that target bacteria, called bacteriophages (phages), may also shape the animal microbiome. Most phage lifecycles can be classified as either lytic or temperate. Lytic phages infect and directly kill bacterial hosts and can directly regulate bacterial population size. Temperate phages, in contrast, have the potential to undergo either a lytic cycle or integrate into the bacterial genome as a prophage. As a prophage, the phage may alter bacterial host phenotypes by carrying novel genes associated with auxiliary metabolic functions, virulence-enhancing toxins, or resistance to other phage infections. Lytic phages may also carry certain auxiliary metabolic genes, which are instead used to takeover bacterial host functions to better accommodate the lytic lifecycle. In either case, the ability to alter bacterial phenotypes may have important ramifications on host-associated communities. This dissertation focused on the genetic contributions that phages, and particularly prophages, provide to the bacterial members of two separate host-associated communities: the honey bee (Apis mellifera) gut microbiome and the amphibian skin microbiome. My second chapter surveyed publicly available whole genome sequences of common honey bee gut bacterial species for prophages. It revealed that prophage distribution varied by bacterial host, and that the most common auxiliary metabolic genes were associated with carbohydrate metabolism. In chapter three, this bioinformatic pipeline was applied to the amphibian skin microbiome. Prophages were identified in whole genome bacterial sequences of bacteria isolated from the skin of American bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), Spring peepers (Pseudacris crucifer) and American toads (Anaxyrus americanus). Prophages were additionally identified in publicly available genomes of non-amphibian isolates of Janthinobacterium lividum, a bacteria found both on amphibian skin and broadly in the environment. In addition to a diverse set of predicted prophages across amphibian bacterial isolates, several Janthinobacterium lividum prophages from both amphibian and environmental isolates appear to encode a chitinase-like gene undergoing strong purifying selection within the bacterial host. While identifying the specific function of this gene would require in vitro isolation and testing, its high homology to chitinase and endolysins suggest it may be involved in the breakdown of either fungal or bacterial cellular wall components. Finally, my fourth chapter revisits the honey bee gut system by investigating the role of geographic distance in bacteriophage community similarity. A total of 12 apiaries across a transect of the United States, from Virginia to Washington, were sampled and honey bee viromes were sequenced, focusing on the lytic and actively lysing temperate community of phages. Although each apiary possessed many unique bacteriophages, apiaries that were closer together did have more similar communities. Each bacteriophage community also carried auxiliary carbohydrate genes, especially those associated with sucrose degradation, and antimicrobial resistance genes. Combined, the results of these three studies suggest that bacteriophages, and particularly prophages, may be contributing to the genetic diversity of the bacterial community through nuanced relationships with their bacterial hosts.
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    The causes and consequences of variation in the cloacal microbiome of tree swallows (Tachycineta bicolor)
    Hernandez, Jessica (Virginia Tech, 2021-08-31)
    Animals are ecological landscapes that host communities of microbes often referred to as microbiomes. These microbes can be transferred between individuals when they come into contact, such as during mating. Microbes that reside in or on any aspect of a host that becomes exposed to the reproductive tract or gametes comprise the reproductive microbiome. These microbes within the reproductive microbiome are important to overall host biology because they can influence host reproductive function, and thus play a role in shaping host ecology, evolution, and fitness. Though previous work has revealed much about the impact of beneficial and pathogenic microbes within the reproductive tract, much is left to be learned from describing the dynamic nature of the reproductive microbiome, and ultimately, how it affects host fitness. For my dissertation, I asked questions regarding how and why reproductive microbiome diversity varies among individuals. For instance, does reproductive microbiome diversity vary with respect to the number of mates or mating activity? Does reproductive microbiome diversity vary with host age or breeding stage? Are there fitness consequences associated with differences in reproductive microbiome diversity? To explore these questions, I studied tree swallows (Tachycineta bicolor), a socially monogamous bird in which both females and males engage in extra-pair mating activity. I focused on the cloacal microbiome as it is the site of contact during mating, and thus where microbes can be sexually transferred between individuals. I found that social partners did not have more similar cloacal microbiome diversity compared to other individuals in the same population, and that cloacal microbiome diversity was similar between sexes (Chapter II). By combining an observational approach with a hormone implant manipulation, I found that neither the number of sires per brood nor the increased mating activity of females significantly influenced cloacal microbiome richness or community structure. However, female age and breeding stage did significantly correlate with cloacal microbiome richness and community structure (Chapter III). Based on these findings, I hypothesized that the effect of mating activity on variation in the cloacal microbiome may only be detectable over a female's lifetime, and not within a single breeding season. In addition, I found evidence for a relationship between lay date and cloacal microbiome structure, after controlling for age. And I found that older females lay earlier in the season compared to younger, first-time breeding females (Chapter IV). These results provide support for a relationship between lay date and the cloacal microbiome and highlight the importance of age to this relationship. Lastly, I discussed future steps that can be taken to extend the framework established by my dissertation research, and thereby gain further insight into the factors shaping the reproductive microbiome (Chapter V).
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    Characterization of fungicide resistance in grape powdery and downy mildew using field trials, bioassays, genomic, and transcriptomic approaches: quinoxyfen, phosphite, and mandipropamid
    Feng, Xuewen (Virginia Tech, 2018-02-06)
    Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere.
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    Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen Batrachochytrium dendrobatidis
    Wax, Noah David (Virginia Tech, 2021-06-22)
    Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.
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    Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera, from the Eastern United States
    Bradford, Emma L.; Wax, Noah; Bueren, Emma K.; Walke, Jenifer B.; Fell, Richard; Belden, Lisa K.; Haak, David C. (Oxford University Press, 2022-12-01)
    Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly.
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    The Curious Poisoned Weed: Poison Ivy Ecology and Physiology
    Dickinson, Christopher Cody (Virginia Tech, 2019-07-11)
    Poison ivy (Toxicodendron radicans (L.) Kuntze) is a native perennial liana widely recognized for the production of urushiol, and the associated contact dermatitis it causes in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of global change. Moreover, poison ivy is an important food source for avian species, and urushiol has numerous applications as a high-value engineering material. Thus, this curious weed has many avenues for future concern, and promise. Here, I address gaps in knowledge about poison ivy ecology and physiology so that we may better understand its weediness and utilize its benefits. I address three core areas: poison ivy establishment patterns; biotic interactions with multiple taxa; and the development of molecular tools for use in poison ivy. I found that the early life stage of seedling emergence is a critical linchpin in poison ivy establishment due largely to herbivore pressure from large grazers. I also describe the multifaceted relationship between poison ivy and avian frugivores that not only disperse the drupes of poison ivy but also aid in reduction of fungal endophytic phytopathogens. A survey of poison ivy urushiols yielded that while variation in urushiol congeners was high across individuals, relative congener levels were stable within individuals over a two month period. Lastly I demonstrate best practices for introducing and transiently expressing recombinant DNA in poison ivy as a step towards future reverse genetic procedures.
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    Developing machine learning tools to understand transcriptional regulation in plants
    Song, Qi (Virginia Tech, 2019-09-09)
    Abiotic stresses constitute a major category of stresses that negatively impact plant growth and development. It is important to understand how plants cope with environmental stresses and reprogram gene responses which in turn confers stress tolerance. Recent advances of genomic technologies have led to the generation of much genomic data for the model plant, Arabidopsis. To understand gene responses activated by specific external stress signals, these large-scale data sets need to be analyzed to generate new insight of gene functions in stress responses. This poses new computational challenges of mining gene associations and reconstructing regulatory interactions from large-scale data sets. In this dissertation, several computational tools were developed to address the challenges. In Chapter 2, ConSReg was developed to infer condition-specific regulatory interactions and prioritize transcription factors (TFs) that are likely to play condition specific regulatory roles. Comprehensive investigation was performed to optimize the performance of ConSReg and a systematic recovery of nitrogen response TFs was performed to evaluate ConSReg. In Chapter 3, CoReg was developed to infer co-regulation between genes, using only regulatory networks as input. CoReg was compared to other computational methods and the results showed that CoReg outperformed other methods. CoReg was further applied to identified modules in regulatory network generated from DAP-seq (DNA affinity purification sequencing). Using a large expression dataset generated under many abiotic stress treatments, many regulatory modules with common regulatory edges were found to be highly co-expressed, suggesting that target modules are structurally stable modules under abiotic stress conditions. In Chapter 4, exploratory analysis was performed to classify cell types for Arabidopsis root single cell RNA-seq data. This is a first step towards construction of a cell-type-specific regulatory network for Arabidopsis root cells, which is important for improving current understanding of stress response.
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    Development of tools to study the association of transposons to agronomic traits
    Yan, Haidong (Virginia Tech, 2020-05-21)
    Transposable elements (Transposons; TEs) constitute the majority of DNA in genomes and are a major source of genetic polymorphisms. TEs act as potential regulators of gene expression and lead to phenotypic plasticity in plants and animals. In crops, several TEs were identified to influence alleles associated with important agronomic traits, such as apical dominance in maize and seed number in rice. Crops may harbor more TE-mediated genetic regulations than expected in view of multifunctional TEs in genomes. However, tools that accurately annotate TEs and clarify their associations with agronomic traits are still lacking, which largely limits applications of TEs in crop breeding. Here we 1) evaluate performances of popular tools and strategies to identify TEs in genomes, 2) develop a tool 'DeepTE' to annotate TEs based on deep learning models, and 3) develop a tool 'TE-marker' to identify potential TE-regulated alleles associated with agronomic traits. As a result, we propose a series of recommendations and a guideline to develop a comprehensive library to precisely identify TEs in genomes. Secondly, 'DeepTE' classifies TEs into 15-24 super families according to sequences from plants, metazoans, and fungi. For unknown sequences, this tool can distinguish non-TEs and TEs in plant species. Finally, the 'TE-marker' tool builds a TE-based marker system that is able to cluster rice populations similar to a classical SNP marker approach. This system can also detect association peaks that are equivalent to the ones produced by SNP markers. 'TE-marker' is a novel complementary approach to the classical SNP markers that it assists in revealing population structures and in identifying alleles associated with agronomic traits.
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    Effects of the antibiotic tetracycline on the honey bee gut microbiome
    Gregory, Casey L. (Virginia Tech, 2024-05-08)
    Host-associated microbial communities, also known as microbiomes, are essential to the health of their hosts, and disturbance of these communities can negatively impact host fitness. The honey bee gut microbiome is a relatively simple host-associated community that makes an excellent model system for studying microbiome stability. In addition, honey bees are essential agricultural pollinators, so factors that impact their health are important for food security. The presented research focused on the stability of the honey bee gut microbiome in response to disturbance from the antibiotic tetracycline. Tetracycline was chosen because it is the most commonly used antibiotic in beekeeping, and may have negative effects on bees through the disruption of their gut microbiomes. The first study presents a new fecal sampling method for studying the honey bee gut microbiome of individual bees over time. This method accurately represented bacterial community structure in the gut microbiome as determined with 16S rRNA gene amplicon sequencing, as fecal and whole gut samples did not differ significantly for individual bees. The fecal sampling technique was then used to examine changes to individual honey bee gut bacterial communities before and after tetracycline exposure. Minimal differences in gut community structure were detected prior to and five days after tetracycline treatment. However, there was variability in how individual gut microbiomes were affected by tetracycline treatment, highlighting the importance of intraspecific variation in response to disturbance. The second study investigated whether the timing of disturbance during a host's life impacts microbiome community stability. Newly emerged bees were treated with tetracycline, returned to their hive, and recollected 7 or 14 days later. The gut communities of the bees were then characterized using 16S rRNA gene amplicon sequencing. Gut microbiome structure of bees treated with tetracycline at emergence differed from controls both 7 and 14 days after emergence, with the antibiotic-treated bees having lower community richness overall. This study showed that early life disturbance of host-associated microbial communities can influence microbiome structure later in life. The final study describes the occurrence of antibiotic resistance genes (ARGs) in honey bee gut bacterial symbionts from hives across the US. Honey bee gut metagenomes were sampled from hives at 13 apiaries located in a transect from Virginia to Washington, and ARG presence was assessed across the sites. We also specifically quantified the abundances of two common tetracycline resistance genes (tet(B) and tet(M)) across apiaries. ARGs, both for antibiotics used in beekeeping and unrelated antibiotics, were detected in honey bee gut bacteria from all apiaries. Tetracycline resistance genes were the most common across all apiaries, and the abundance of two tetracycline resistance genes varied by apiary. Members of the honey bee gut microbiome contained different proportions of ARGs, but taxa within a single family contained similar proportions, possibly indicating phylogeny plays a role in ARG accumulation. In particular, Gilliamella and Frischella, both in the family Orbaceae, contained the highest percentages of ARGs. The results from this study suggest honey bee bacteria act as reservoirs of ARGs. Overall, the presented research contributes to the field of biology by highlighting the importance of intraspecific variation in host-associated microbial communities and presenting a new method for studying honey bee gut microbiome variation at the individual-level, showing that early life events in honey bees influence microbiome development, and suggesting that honey bee bacterial symbionts have adapted to deal with antibiotic disturbance through the accumulation of ARGs.
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    Epigenetic Responses of Arabidopsis to Abiotic Stress
    Laliberte, Suzanne Rae (Virginia Tech, 2023-03-17)
    Weed resistance to control measures, particularly herbicides, is a growing problem in agriculture. In the case of herbicides, resistance is sometimes connected to genetic changes that directly affect the target site of the herbicide. Other cases are less straightforward where resistance arises without such a clear-cut mechanism. Understanding the genetic and gene regulatory mechanisms that may lead to the rapid evolution of resistance in weedy species is critical to securing our food supply. To study this phenomenon, we exposed young Arabidopsis plants to sublethal levels of one of four weed management stressors, glyphosate herbicide, trifloxysulfuron herbicide, mechanical clipping, and shading. To evaluate responses to these stressors we collected data on gene expression and regulation via epigenetic modification (methylation) and small RNA (sRNA). For all of the treatments except shade, the stress was limited in duration, and the plants were allowed to recover until flowering, to identify changes that persist to reproduction. At flowering, DNA for methylation bisulfite sequencing, RNA, and sRNA were extracted from newly formed rosette leaf tissue. Analyzing the individual datasets revealed many differential responses when compared to the untreated control for gene expression, methylation, and sRNA expression. All three measures showed increases in differential abundance that were unique to each stressor, with very little overlap between stressors. Herbicide treatments tended to exhibit the largest number of significant differential responses, with glyphosate treatment most often associated with the greatest differences and contributing to overlap. To evaluate how large datasets from methylation, gene expression, and sRNA analyses could be connected and mined to link regulatory information with changes in gene expression, the information from each dataset and for each gene was united in a single large matrix and mined with classification algorithms. Although our models were able to differentiate patterns in a set of simulated data, the raw datasets were too noisy for the models to consistently identify differentially expressed genes. However, by focusing on responses at a local level, we identified several genes with differential expression, differential sRNA, and differential methylation. While further studies will be needed to determine whether these epigenetic changes truly influence gene expression at these sites, the changes detected at the treatment level could prime the plants for future incidents of stress, including herbicides.
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    Evaluating the Adaptive Genomic Landscape of Remnant and Backcross American Chestnut Populations to Inform Germplasm Conservation
    Sandercock, Alexander M. (Virginia Tech, 2023-07-27)
    The American chestnut tree (Castanea dentata) is a deciduous tree that largely exists in the eastern United States along the Appalachian Mountain range. Approximately 100 years ago, a fungal pathogen (Cryphonectria parasitica) decimated chestnut populations, resulting in the loss of billions of trees. Disease-resistant American chestnut populations have been developed, but the introgression of wild adaptive diversity into these breeding populations will be necessary to develop locally adapted and disease resistant chestnut trees for reintroduction. In this dissertation, I presented our findings which addressed previous gaps in knowledge regarding the population genomics of wild and backcross American chestnut populations. I 1) estimated the genomic diversity, population structure, and demographic history of remnant wild American chestnut populations; 2) revealed the genomic basis of local climate adaptation in American chestnut, developed a novel method to make tree sampling estimates for germplasm conservation, and defined unique seed zones for American chestnut based on climate and genotype, and 3) determined the amount of wild adaptive diversity captured by the backcross breeding program and made recommendations for their replanting region. These results will inform the development of a breeding plan for the introgression of adaptive diversity into backcross and transgenic chestnut populations.
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    Evaluation of Genomic Prediction and the Agronomic Performance of Facultative Barley
    Reith, Francis Arthur (Virginia Tech, 2024-10-28)
    Cultivated barley typically exhibits either a winter growth habit or a spring growth habit. Some cultivars display a facultative growth habit, allowing them to be cultivated as either winter or spring crops. This study evaluated 1,128 elite barley cultivars and breeding lines under fall and spring sowing to determine which lines had a facultative growth habit and the underlying genetics thereof. In the fall of 2021 and subsequent spring of 2022, The first trial focused on identifying genetic factors associated with facultative habit. Results indicated that facultative lines were rare, with the majority exhibiting a winter growth habit. GENOME WIDE SCANS revealed no novel QTL associated with facultative habit, but significant QTLs on chromosome 4H were identified, correlating with the vernalization gene "VRN-H2." Several haplotypes found on chromosome 4H within appear significant and may contribute to differences in facultative habit. Only 28% of facultative lines could be accurately predicted based on genetic data, suggesting that facultative habit is a more complex trait than previously understood. Significant epistatic interactions between chromosome 4H and 4 other chromosomes were discovered. These findings indicate facultative habit is a much more quantitative trait than previously reported. The second trial involved growing the best-performing lines from the first trial under both fall and spring sowing conditions. Winter-sown barley consistently outperformed spring-sown barley in grain yield across all facultative lines. Despite strong performance under spring conditions, yield rankings were inconsistent across both sowing seasons, implying that agronomic performance cannot be reliably predicted across seasons. Notably, the Virginia Tech malt barley line 'Avalon' demonstrated facultative growth but exhibited poor agronomic quality under spring sowing. In contrast, lines such as 'VA22M-20DH1349' and 'VA22M-20DH1182' showed superior performance in both sowing regimes, indicating their potential for future breeding programs and agronomic trials for facultative barley in the American East.
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    Evaluation of integrated weed management techniques and their nuances in Virginia crop production
    Beam, Shawn Christopher (Virginia Tech, 2019-11-04)
    Herbicide resistant weeds are driving implementation of integrated weed management (IWM). A new tactic to manage weeds is harvest weed seed control (HWSC), which targets weed seeds retained on the plant at crop harvest and either destroys, removes, or concentrates them. Research is limited on the effectiveness of HWSC in US cropping systems. For HWSC to be effective it is important to know when and how many seed are shed from a weed species in relation to crop harvest. Research was conducted to quantify when weed seed are shattered from 6 economically important weed species, four broadleaf (redroot pigweed, common ragweed, common lambsquarters, and common cocklebur) and two grass species (large crabgrass and giant foxtail). Results indicate that among summer annuals, broadleaf species retain larger proportions of their seed compared to grass species at the first opportunity for soybean harvest. As harvest was delayed, more seeds shattered from all species evaluated, indicating timely harvest is critical to maximizing HWSC effectiveness. Studies were conducted on grower fields in Virginia to evaluate the effectiveness of HWSC (field residue and weed seed removal). Results indicate that HWSC can significantly reduce populations of Italian ryegrass in wheat and common ragweed in soybean in the next growing season, but reductions were not observed for Palmer amaranth in soybean. Investigating IWM system for common ragweed control in soybean, HWSC was found to be less effective than soybean planting date (i.e. double cropping after wheat) at reducing common ragweed populations. However, the effectiveness of HWSC varied by location. If HWSC adoption were to become widespread, weeds could adapt by shedding seed earlier in the season. Research was conducted by growing Palmer amaranth populations from across the eastern US in a common garden. Currently there are differences in flowering time and seed shatter among Palmer amaranth populations based on the location of the maternal population, indicating potential for adaptation. This research demonstrates that HWSC is a viable option for weed management in US cropping systems but needs to be stewarded like any other weed management tool.
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    Evaluation of Seed Impact Mills for Harvest Weed Seed Control in Soybean and Wheat in the Eastern United States
    Russell, Eli Carnley (Virginia Tech, 2024-11-11)
    Harvest Weed Seed Control (HWSC) concentrates, removes, or destroys weed seeds as they pass through the combine. Seed impact mills are modifications that are mounted directly to the back of a combine and are one way to implement HWSC. Seed impact mills kill weed seeds during harvest, preventing seeds from being added to the soil seedbank. Mills like the Redekop Seed Control Unit (SCU) and the integrated Harrington Seed Destructor (iHSD) could be used in soybean and wheat production in the eastern United States. Understanding the effectiveness and limitations of these mills is important for grower adoption. So, the aim of this research was to evaluate the efficacy of two seed impact mills, the Redekop SCU and the iHSD, in soybean and wheat. The first objective tested general seed kill of problematic species in soybean and wheat and seed kill in adverse conditions, such as high chaff flow rate into the mill and high chaff moisture. Results from objective one indicate that both the Redekop SCU and iHSD killed >98% and >91% of problematic weed seeds in soybean and wheat, respectively. Increases in chaff flow rate and chaff moisture resulted in a decrease in seed kill for specific species depending on the mill. But even at high chaff flow rates, seed kill remained >98% and >77% in soybean and wheat, respectively. At high chaff moisture, seed kill remained >98% and >74% in soybean and wheat, respectively. The second objective evaluated the percentage of weed seeds that bypassed the seed impact mill by exiting the combine in the straw fraction and the percentage of weed seeds that were killed when they entered the seed impact mill during harvest with a commercial combine. Results at field scale indicated that <5% of weed seeds bypassed the seed impact mill by exiting the combine in the straw fraction during harvest in soybean and wheat. Additionally, during a commercial harvest, the seed impact mills killed >99% and >89% of seeds in soybean and wheat, respectively. The third objective monitored population density changes for common ragweed (Ambrosia artemisiifolia) in soybean and Italian ryegrass (Lolium perenne ssp. multiflorum) in wheat following a harvest with a seed impact mill. Results from objective three indicated that in the growing season following a harvest with a seed impact mill, common ragweed density was reduced by 26% and 77% in the spring and fall, respectively, in soybean, and Italian ryegrass density was reduced by 48% in wheat. The fourth objective evaluated Palmer amaranth (Amaranthus palmeri) and its ability to shift its flowering timing in response to HWSC. If weeds flower earlier, they could shatter seeds earlier, and those seeds would bypass HWSC. Through selective breeding, two populations of Palmer amaranth experienced a shift in flowering timing such that the third generations flowered 54.7 and 41.0 days sooner in the greenhouse than the initial generations. In a common garden experiment, the second generations flowered 5.5 and 8.9 days sooner than the initial generations. These results indicate that seed impact mills, like the Redekop SCU and iHSD, can deliver high seed kill rates to a range of weed species at commercial scale in both soybean and wheat. Even in adverse conditions, the mills still killed >74% of seed from tested species. However, weed species can adapt to HWSC selection pressures, resulting in a loss of HWSC efficacy. Overall, this research indicates that seed impact mills are a good tool that growers can implement to reduce the number of weed seeds being returned to the soil seedbank.