Browsing by Author "Wrighton, Kelly, committee member"
Now showing 1 - 12 of 12
Results Per Page
Sort Options
Item Open Access Combined effects of warming and drying on a temperate-to-boreal forest ecotone exert additive changes on soil microbiome structure and diversity(Colorado State University. Libraries, 2020) Dean, Daniel, author; Trivedi, Pankaj, advisor; Leach, Jan E., committee member; Wrighton, Kelly, committee member; Reich, Peter B., committee memberThe soil microbial community is an important mediator of many ecosystem functions, so understanding dynamics under climate change. These responses could be more robust in transitional zones such as the temperate-to-boreal forest ecotones, which are poised to experience substantial changes under projected climate change over the next century and beyond. Because these systems are projected to move towards a warmer, drier climate, it is important to understand how the soil microbiome's structure and interactions shift under such conditions. Here, we examined the response of microbial communities to simulated warming and drought conditions using the B4WarmED (Boreal Forest Warming in an Ecotone in Danger) experiment in Minnesota, USA. B4WarmED is a fully factorial blocking experiment which uses in situ experimental 3.4°C warming and precipitation reduction to simulate the projected regional late-21st century climate. Using Shannon-Weaver Diversity and Canonical Analysis of Principled Coordinates, we found that combined warming and drying effects exerted significant effects on the diversity and structure of microbial communities after 8 years of warming, and 5 of drought treatments. Specifically, warming and drying effects appeared to combine additively, rather than exhibiting nonlinear interactive effects, at the community level. Per-taxon linear models revealed a sizeable portion of individual microbes exhibit a significant abundance response to one or both of warming and drying effects. However, co-occurrence network analysis and Dufrene-Legrende Indicator Value characterization revealed a smaller portion of bacterial sub-communities with persistent taxonomical makeup and response profiles across treatments. Within the microbial communities our analysis identified three types of taxon-specific responses to climate change stressors: resistant, opportunistic, and sensitive, with most taxa being resistant to warming and drying effects. However, our results provide strong evidence that combined warming and drought influences will impact soil microbial communities of temperate-to-boreal ecotone forests ("boreal ecotone" hereafter), with potential implications for ecosystem functioning.Item Open Access Effects of plant-selected rhizobacterial communities on the drought resistance of tomato plants(Colorado State University. Libraries, 2021) Monohon, Samantha J., author; Vivanco, Jorge M., advisor; Manter, Daniel, committee member; Wrighton, Kelly, committee memberDrought stress has had devastating effects for vegetable growers world-wide, leading to much recent research focusing on the development of drought-resilient crops. The importance of the rhizosphere microbiome in plant performance under drought stress is under development, including the use of beneficial inoculations of PGPR and transplanting of microbial communities. However, further research is needed to fully understand plants' innate abilities in mediating rhizobacterial recruitment to benefit plant resistance to drought stress. Here, two greenhouse studies were performed to determine the efficacy of conditioned soils containing plant-selected rhizobacterial communities as a means to increase drought resilience of host plants. Soils were autoclaved to lower microbial complexity and ensure the greatest plant influence over soil rhizobacterial recruitment. Tomato plants were grown in soils, autoclaved and control, to assess microbial recruitment under a gradient of water treatments: well-watered, moderate drought and severe drought. Autoclaved soils revealed a potential amplification of plant-selective influence over microbial community assemblage for drought-specific bacteria. Inoculants derived from this study were used to observe the impacts of microbial history on a plant's ability to tolerate contemporary drought stress conditions. Microbial history was shown to have a significant effect on microbial community composition and plant performance under drought conditions. To further apply the conditioned effects of microbial communities on tomato plants under severe drought stress, a multi-generational study was performed to amplify plant-selected microbial communities from soils previously exposed to severe drought treatment. Effects of soil conditioning and microbial history suggested the presence of bacteria, conditioned over generations of plant-selection, involved in microbially-mediated plant growth restriction of tomatoes as a drought avoidance strategy. In summary, prior exposure of plants and microbial communities to drought stress may provide beneficial traits for host plants under contemporary drought conditions.Item Open Access Evaluation of cover crops in reducing Spongospora subterranea inoculum through qPCR and microscopy assays(Colorado State University. Libraries, 2021) Alaryan, Maryam M., author; Charkowski, Amy, advisor; Stewart, Jane, committee member; Wrighton, Kelly, committee memberCover crops have been used for years as an effective practice to manage soil-borne pathogens. Beside their other beneficial properties in enhancing soil quality and fertility, they can suppress soil-borne pathogens and reduce their populations in the soil. Spongospora subterranea is a soil-borne obligate biotrophic plasmodiophorid that causes powdery scab on potato tubers and gall formation on roots. Powdery scab tuber lesions are filled with resting spores (sporosori) and reduce potato quality and marketability. S. subterranea also vectors Potato mop-top virus, which causes necrotic arcs and spraing in tuber flesh. Currently, there are no effective methods to manage S. subterranea, which has a wide host range in addition to potato. This study was conducted to determine whether cover crops can decrease S. subterranea population levels in the soil. Eighteen cover crops, including legumes and mustards were assessed, and for each plant line, five plants were grown in individual pot that was inoculated with 10 sporosori/g of potting mix by comparison with five plants that were grown in non-inoculated potting mix. After harvest, plant roots were stained using trypan blue and examined under the light microscope, and qPCR was performed to determine S. subterranea inoculum level in the potting mix. The results suggested that S. subterranea invaded all the cover crop roots; however, the pathogen was unable to complete its life cycle on eleven out of eighteen cover crops based on post-harvest qPCR results. To confirm our results, a follow up experiment was conducted by inoculating the potting mix with 40 sporosori/g to better detect the pathogen, in addition to autoclaving the peat moss prior to planting. The roots were stained with DAPI after harvest and examined under the fluorescence microscope and S. subterranea DNA levels was quantified in the eleven cover crops by qPCR. The results showed that buckwheat, barley, and legumes have the potential to stop the pathogen from increasing in the soil where there was no significant increase in the inoculated samples in most of the tested cover crops. The results from this experiment will be used to guide in-field cover crop experiments and to advise farmers on cover crops that may not increase the inoculum levels of S. subterranea in the soil.Item Open Access Exploring interactions among biological soil crusts, plant germination, and morphological seed traits: implications for plant community assembly and dryland restoration(Colorado State University. Libraries, 2023) Bacovcin, John, author; Havrilla, Caroline, advisor; Ocheltree, Troy, committee member; Wrighton, Kelly, committee member; Paschke, Mark, committee memberArid and semi-arid (dryland) ecosystems make up over 40% of our plant's terrestrial surface and are incredibly vulnerable to land degradation. To combat dryland degradation, active plant and soil restoration is often needed and the role of plant-soil microbe interactions can be key to dryland restoration trajectories. Within drylands, biological soil crusts (biocrusts), collections of cyanobacteria, algae, lichen, and moss are key surface communities that influence soil processes (e.g., stability, nutrient cycling, hydrology) and can thereby strongly influence recruitment of dryland plants. These biocrusts may interact with plant functional traits (i.e., seed morphological traits), and these interactions can influence germination. However, much is still unknown about mechanisms that underlie these interactions and how plant functional traits mediate effects of biocrusts on plant germination. To investigate these knowledge gaps, I conducted two studies: (Chapter 1) a global meta-analysis of the role of morphological seed traits in determining biocrust effects on germination, and (Chapter 2) a full-factorial greenhouse study examining the effects of biocrust inoculum cover treatments and plant functional traits on plant recruitment to investigate questions about how biocrust heterogeneity and biotic components of biocrusts in the context of restoration. To explore effects of morphological seed traits on plant germination responses to biocrusts (Ch. 1), we compiled a global database of 491 studies of biocrust effects on plant germination encompassing 101 unique plant species and their associated morphological seed traits. For the greenhouse study (Ch. 2) we seeded two seed mixes on three different inoculum cover treatments (i.e., 0%, 30%, and 100%) using both biologically active (live) and autoclaved biocrust inoculum, to assess effects of cover heterogeneity, biological biocrust activity, and plant functional traits on percent germination. Results from the meta-analysis showed that morphological seed traits do mediate plant germination responses to biocrusts, and that, in general, germination of smaller seeded species with appendages was increased by biocrusts. Results from the greenhouse study showed that, in a restoration context, increasing cover of biocrust inoculum increases plant germination, and that these effects were explained by physical rather than biotic effects of inoculum on germination. As in Chapter 1, we found that biocrusts effect on germination differed across plant functional groups and that seed traits also influenced germination responses to biocrust inoculum cover treatments. Together, both studies showed that morphological seed traits mediate effects of biocrusts on plant germination. These findings increase understanding of the role of biocrusts in determining dryland plant community assembly and have implications for dryland restoration.Item Open Access Intra and inter-annual patterns in Lake Yojoa, Honduras: disentangling biogeochemical drivers of a tropical monomictic lake ecosystem(Colorado State University. Libraries, 2022) Fadum, Jemma Mazuri, author; Hall, Ed, advisor; Wrighton, Kelly, committee member; Ross, Matt, committee member; Burgin, Amy, committee memberTo view the abstract, please see the full text of the document.Item Open Access Investigating the relationship between cover crop species diversity, composition and function of the soil microbiome(Colorado State University. Libraries, 2023) Seitz, Valerie, author; Prenni, Jessica, advisor; Wrighton, Kelly, committee member; Schipanski, Meagan, committee member; Nishimura, Marc, committee memberCropping diversification, such as cover cropping, can contribute to sustainable agriculture by enhancing soil health and promoting ecosystem services through interactions with the soil microbial community. One important mechanism through which cover crops impact soil health is via root exudation, the release of organic compounds from plant roots into the soil region surrounding the roots, the rhizosphere. Root exudation varies among cover crop species, growth stages, and edaphic and environmental conditions resulting in a myriad of effects on the rhizosphere. Plant-derived inputs, like root exudates, modulate the soil microbial community, influencing microbial biomass, community structure, and catalyzing biogeochemistry. As a result, cover crops are linked to microbial changes that impact soil nutrient cycling and organic matter decomposition leading to a legacy impact on primary crop yield and health. Understanding the intricate relationship between cover crop root exudation composition and the soil microbiome is crucial for optimizing cover crop selection, management practices, and harnessing cover crops for precision microbiome management in agroecosystems. My dissertation demonstrates that cover crop root exudation differs considerably across cover crop species, and cultivars within species, and reveals cover crop metabolic impacts on soil microbial composition and function, which play a large role in the generation and maintenance of healthy soils to support our agricultural needs.Item Open Access Metabolic engineering interventions for sustainable 2,3-butanediol production in gas fermenting Clostridium autoethanogenum(Colorado State University. Libraries, 2023) Ghadermazi, Parsa, author; Chan, Siu Hung, advisor; Wrighton, Kelly, committee member; Reisfeld, Brad, committee memberGas fermentation provides a promising platform to turn low-cost and readily available single-carbon waste gases into commodity chemicals such as 2,3-butanediol. Clostridium autoethanogenum is usually used as a robust and flexible chassis for gas fermentation. Here, we leveraged on constraints-based stoichiometric modeling and kinetic ensemble modeling of the C. autoethanogenum metabolic network to provide a systematic in silico analysis of metabolic engineering interventions for 2,3-butanediol overproduction and low carbon substrate loss in dissipated CO2. Our analysis allowed us to identify and to assess comparatively the expected performances for a wide range of single, double, and triple interventions. Our analysis managed to individuate bottleneck reactions in relevant metabolic pathways when suggesting intervening strategies. Besides recapitulating intuitive and/or previously attempted genetic modifications, our analysis neatly outlined that the interventions - at least partially - impinging on by-products branching from acetyl-CoA and pyruvate (acetate, ethanol, amino acids) offer valuable alternatives to the interventions focusing directly on the specific branch from pyruvate to 2,3-butanediol.Item Embargo Of microbes and mothers: evaluating the complex maternal-neonatal interaction and microbiome-immunity development with novel Lactobacillus vaccination(Colorado State University. Libraries, 2024) Ecton, Kayl E., author; Abdo, Zaid, advisor; Dean, Gregg, advisor; Wrighton, Kelly, committee member; Vilander, Allison, committee member; Argueso, Lucas, committee memberThe task of identifying an optimal vaccination strategy for neonates has been challenging scientists and physicians alike. Multiple factors contribute to the difficulty in establishing an optimal platform including the complexity of the maternal-fetal dyad, a neonatal Th2 skewed profile and the role of the parallel development of the immune system and the gut microbiome (8). Disease remains a main cause of infant morbidity and mortality, encouraging the discovery of novel infant vaccinations to be delivered during the first 28 days of life to provide protection (41). Passive protection from the maternal transfer of transplacental IgG and both IgG and IgA in breastmilk has a limited window of operation, leaving the maturing neonate at risk (128). Although exact mechanisms remain to be elucidated, here we examine the complex crosstalk between mother-fetus and maternal-neonate dyads, neonatal microbiome-immunity development, and optimal delivery strategies for neonatal vaccine development. In this dissertation we investigated the role of maternal infection prior to gestation, neonatal challenge after vaccination, and vaccine effectiveness after exposure to virus. We evaluated the use of a novel vaccine platform developed previously in the lab as an orally delivered mucosal targeting subunit vaccine in Lactobacillus acidophilus. We investigated the effectiveness of the recombinant vaccine with and without adjuvants in a neonatal experimental design model and discovered increased virus specific responses in neonates vaccinated with adjuvants when challenged with rotavirus. We show a significant impact of maternal influence on neonatal outcomes. Beyond the immunogenic strength of the novel Lactobacillus acidophilus vaccine platform in neonates, we identified induced shifts to the gut microbial communities that occurred with vaccination or infection. We saw a shift in the gut microbiome over the course of a 7-day rotavirus challenge in neonates that did not return to baseline during the observation period, even after no virus shedding was detected in fecal samples. We also evaluated the impact of different doses, 1x106 CFU/dose and 1x109 CFU/dose, on the immune response and the gut microbiome. We confirmed the role of fecal microbiome transplants in breeding does to normalize for the maternal microbiome prior to gestation. Our results indicate that there are modifications to the gut microbiome and changes in immune antibodies during vaccination and infection. While we did not pursue a specific mechanism crosslinking the maternal-neonatal interaction and the gut-immunity relationship, we do consider the presence of such a connection.Item Open Access The effects of irrigation retirement on soil carbon dynamics of a continuous maize agroecosystem(Colorado State University. Libraries, 2023) Mendoza-Martinez, Violeta, author; Schipanski, Meagan, advisor; Wrighton, Kelly, committee member; Prenni, Jessica, committee memberOver half of the world's fresh water is used in crop production and, in some key agricultural regions, use far exceeds local water availability and recharge rates. With the increasing strain on freshwater resources caused by climate change and a growing population, agriculture is under pressure to reduce its water consumption and large areas of currently irrigated farmland across the Western U.S. will likely transition into dryland agriculture over the coming decades. The effects this will have on global soil carbon (C) dynamics, however, remain unclear. In 2016, a study was established in Northern Colorado to understand how stopping irrigation affects soil C turnover in a no-till, continuous maize agroecosystem. Earlier results showed limited responses of the soil microbial community to irrigation retirement, but differences in soil heterotrophic respiration (Rh) rates were detected after two years of accumulated differences in plant residue inputs, thus suggesting a possible co-limitation of water and available C to the microbial community. We continued this experiment through 2022 to further explore the relationship between soil moisture and C inputs in shaping the soil microbial community under the new watering regimes and the consequential effects on soil respiration (Rs) as an indicator of soil organic C (SOC) turnover rates. Two seasons of data collection in 2021 and 2022 showed decreases in available soil water, bacteria, fungi, protozoa and actinomycetes fatty acid methyl ester (FAME) biomarkers, activities of four extracellular enzymes and soil autotrophic respiration in response to both reductions in irrigation and plant inputs, with strong interactive effects between the two factors. However, plots under dryland conditions had higher concentrations of dissolved organic carbon (DOC) and muted differences in soil Rh when compared to their irrigated counterparts; differences in Rh between fallow treatments with (YF) and without residue inputs (LTF), on the other hand, were more pronounced. Soil Rs in fallow plots was consistently, positively correlated with field soil temperature, while correlations with moisture were weak or even negative, thus suggesting soil moisture was not a strong direct driver of Rh. We investigated the direct and indirect influences of variables collected monthly across two seasons on soil Rh to test our hypothesized model using structural equation modeling. In contrast to the cumulative treatment level impacts of plant inputs and irrigation, monthly soil moisture measurements had a stronger, direct effect on Rh than substrate availability as estimated by water-extractable DOC. The final model only explained 24% of the variability in soil Rh. Changes in global C dynamics can be expected with transition of land areas from irrigated to dryland agriculture. However, focusing on soil health, resource conservation practices and the resiliency of the soil microbiome can be the key to minimize the potential negative impacts of this transition.Item Open Access The microbiome surrounding death and decay: microbial ecology of food processing, meat spoilage, and human decomposition environments(Colorado State University. Libraries, 2021) Belk, Aeriel D., author; Metcalf, Jessica L., advisor; Martin, Jennifer, committee member; Wrighton, Kelly, committee member; Ben-Hur, Asa, committee memberTo view the abstract, please see the full text of the document.Item Open Access Tracking the impact of wildfire on the soil microbiome across temporal scales(Colorado State University. Libraries, 2024) Nelson, Amelia Rose, author; Wilkins, Michael J., advisor; Hall, Ed, committee member; Borch, Thomas, committee member; Rhoades, Charles, committee member; Wrighton, Kelly, committee memberAs climate change progresses, the western United States is experiencing shifting wildfire behavior to more frequent and severe wildfires. Wildfires reduce soil microbial biomass and alter the soil microbiome community composition, selecting for "pyrophilous" microbial taxa with encoded traits that enable them to persist during wildfire or thrive in the soil thereafter. The soil microbiome is a key player in ecosystem carbon (C) cycling through the mediation of soil organic matter decomposition and stabilization. In addition to post-fire shifts in the soil microbiome, wildfire decreases soil C pools through combustion and alters C quality via fire-induced transformations to aromatic pyrogenic C (PyC). The intricate interplay between wildfire-induced alterations to soil microbiome composition and function, and subsequent ecosystem C cycling, remains poorly understood across different temporal and spatial scales. Leveraging multi-omics data alongside soil chemistry information (e.g., mass spectrometry) can offer insights into how shifting wildfire behavior may influence microbially mediated C cycling in forest ecosystems across the western US. To address this knowledge gap, I developed an extensive multi-omic dataset from burned Colorado subalpine coniferous forest soils collected over time (spanning 1 to 60 years following burning) and disturbance severity (low and high fire severity). This dataset includes 108 metagenomes and 12 metatranscriptomes, resulting in 1651 metagenome-assembled genomes (MAGs) that represent many of the dominant putative pyrophilous taxa previously identified in compositional studies. This dissertation presents the key findings derived from this comprehensive dataset, with the primary goal of addressing how wildfire impacts the soil microbiome with a focus on microbial interactions with soil C. Chapter 1 serves as a comprehensive literature review, providing an overview of prior research relevant to the research presented thereafter. It underscores the timely relevance of this dissertation research by examining how wildfire behavior is shifting globally with climate change and anthropogenic forcing. Given the critical role of forest ecosystems as significant global C sinks, understanding the repercussions of wildfires on ecosystem biogeochemistry is imperative. I broadly summarize previous research regarding severe wildfire impacts to soils and the soil microbiome and focus on existing knowledge gaps regarding the function of the post-wildfire soil microbiome across differing burn severities and time since fire. In Chapter 2, I characterize how burn severity impacts the soil microbiome one year post-fire in Colorado (CO) subalpine coniferous forests using soil samples collected in July 2019 from within the 2018 Ryan and Badger Creek fire burn scars that represent a burn severity gradient (control, low, moderate, and high severity burned soils). I used a suite of tools to understand both the impacts to soil chemistry and the soil microbiome, including Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to characterize dissolved soil organic matter, 16S rRNA gene and ITS amplicon sequencing for soil microbiome composition, and coupled metagenomics and metatranscriptomics to identify shifts in soil microbial functional potential. The combination of these tools allowed me to characterize the entire soil microbiome, including bacteria, fungi, and viruses. From metagenomic sequencing, I recovered 637 MAGs, 1982 unique DNA and RNA viral populations, and 2 fungal genomes from low and high severity burned soil samples. I broadly found that Actinobacteria dominated the fraction of enriched and active bacterial taxa within high severity surficial soils and exhibited traits (e.g., heat resistance, fast growth, expression of genes for degrading aromatic PyC) that enabled them to survive the soil heating and thrive after the disturbance. Ectomycorrhizal fungi (EMF), key symbionts of coniferous trees and other plant taxa, were depleted in severely burned soils. Lastly, there were abundant viruses targeting dominant Actinobacteria MAGs that likely played important roles in assembly of the post-wildfire soil microbiome and serve as top-down controls of C cycling within the system. Overall, this study served as a holistic and comprehensive snapshot of the post-wildfire soil microbiome at one point in time and laid the foundation for forming hypotheses and guiding the subsequent studies. Building upon the groundwork laid in Chapter 2, Chapter 3 broadly evaluates the relative importance of putative pyrophilous traits identified between one year and 11 years following wildfire. Additionally, I explored the applicability of other proposed conceptual life history strategy frameworks (e.g., Y-A-S framework) in defining post-wildfire soil microbial dynamics. I utilized a series of soil samples collected from a chronosequence of CO wildfire burn scars representing 1, 3, 5, and 11 years following low- and high-severity wildfire. Using genome-resolved metagenomic approaches and combining this newly generated MAG catalog with the MAGs reconstructed from sequencing in Chapter 1 resulted in a total of 825 bacterial MAGs. Again, this dataset was coupled to various soil chemistry datasets, microbial biomass measurements (via PLFA), and marker gene sequencing data. I found that the potential for fast growth was an important bacterial trait driving dominance in the post-wildfire soil microbiome for up to approximately 11 years post-fire. Moreover, I observed that MAGs investing in traits aimed at acquiring diverse resources from the external environment often dominated severely burned soils, aligning with the 'A' strategy outlined in the Y-A-S framework. These insights suggest that microbial trait profiles play a pivotal role in shaping post-wildfire soil microbial successional dynamics. Furthermore, the study marks a significant step towards unraveling how trait-based frameworks can offer valuable insights into post-disturbance microbial ecology. In Chapter 4, the focus shifts to investigating one of the most extreme scenarios that can occur in a terrestrial ecosystem with severe wildfire: a burning-induced aboveground vegetation shift. Pile burning is a common fuel reduction or site preparation practice wherein logging residue is burned on the forest floor and, because of the high soil temperatures often reached during pile burning, can serve as a surrogate for studying impacts to soil caused by severe wildfire. Following clear-cut harvesting, pile burning can lead to the creation of persistence openings dominated by herbaceous plants within successfully regenerating conifer forest. In this study, a paired 60-year chronosequence of burn scar openings and surrounding forests that regenerated after clear-cut harvesting provided a unique opportunity to study soil microbiome changes associated with two distinct ecosystem development trajectories (i.e., burning-induced aboveground vegetation shift, regenerating coniferous forest). The primary objective was to identify whether the belowground soil microbiome exhibited resilience to a disturbance-induced aboveground vegetation shift. I collected soils from the aforementioned chronosequence and interrogated soil microbiome composition (via marker gene sequencing), functional potential (via metagenomics), and function (via laboratory incubations). There were compositional shifts in the soil microbiome that mirrored the ongoing aboveground vegetation shifts, with short-term changes to microbial community composition and C cycling functionality closely resembling a post-wildfire soil microbiome (e.g., PyC degradation). However, over the six-decade chronosequence the soil microbiome composition and function both displayed resilience, converging with that of the surrounding regenerating forest. This final research chapter extended the findings from the previous studies by exploring the longevity of wildfire impact to the soil microbiome in the extreme case of a burning-induced aboveground vegetation shift. The final chapter (Chapter 5) summarizes the key findings of this doctoral research and discusses potential research implications and applications along with future research directions and remaining knowledge gaps. In summary, the aims of this dissertation research were to identify how burn severity influences the soil microbiome composition and function one year post-fire (Chapter 2), assess the longevity of these impacts and the applicability of conceptual traits-based frameworks to the post-fire soil microbiome (Chapter 3), and evaluate the resilience of the belowground soil microbiome to a burning-induced multidecadal aboveground vegetation shift (Chapter 4). This research significantly advances our understanding of the impacts of wildfires on crucial forest ecosystems, with a specific emphasis on ecosystem C cycling.Item Open Access Unlocking sorghum adaptive potential through investigations into pleiotropic control of chilling tolerance by Tannin1(Colorado State University. Libraries, 2023) Schuh, Anthony, author; Morris, Geoffrey, advisor; Argueso, Cristiana, committee member; Wrighton, Kelly, committee memberChilling tolerant crops can positively impact agricultural sustainability through lengthened growing seasons and improved water and nitrogen use efficiency. In sorghum (Sorghum bicolor [L.] Moench), the fourth most grown grain, coinheritance of qSbCT04.62, the largest effect chilling tolerance locus, with Tannin1, the major gene underlying undesirable grain proanthocyanidins, has stymied breeding for chilling tolerance. To investigate the genetic basis of qSbCT04.62, including its coinheritance with Tan1, we developed near isogenic lines (NILs) with chilling tolerant haplotypes around qCT04.62. In the first study we genotype the NILs and investigate the introgressions physiological control over the cold stress response. Genome sequencing revealed that the CT04.62+ NILs introgressions on chr04 include Tannin1, a homolog of Arabidopsis cold regulator CBF, peak SNPs for qCT04.62 from multi-family NAM, and 61.2-62 Mb of HKZ ✕ BTx623 NAM family qCT04.62 confidence interval. Grain tannins were correlated with Tan1 genotype, revealing heterogeneity in one NIL at Tannin1. Controlled environment chilling assays found no genotype by environment interaction on growth by chilling per se in parents or NILs. Cold germination was reduced at 15°C and superior at 20 and 25°C in the chilling tolerant parent compared to chilling sensitive, but unchanged between NILs. The introgression also did not regulate a chilling induced increase in non-photochemical quenching. In the second study we investigated Tan1 function with a transcriptome analysis of the NIL's response to chilling stress. Tannin1 was widely expressed in sorghum tissues but did not promote a transcriptional response in chilling tolerance related molecular pathways including lipid remodeling, phytohormone signaling, CBF upregulation, photoprotection, and ROS mitigation. GO analysis also found no significant term enrichments at the p < 0.1 threshold. Only 17 genes had expression patterns regulated by polymorphisms in the introgressions, seven cis, and ten trans, with little evidence of co-regulation. Further, Tannin1 was functionally divergent from its Arabidopsis ortholog TTG1 and other WD40 orthologs in regulating leaf anthocyanin biosynthesis. Overall, these findings suggest that linkage, not pleiotropy, underpins the coinheritance of Tan1 and CT04.62+, unlocking the use of CT04.62+ for sorghum improvement. Further, these results imply a lack of deleterious fitness effects of tan1 alleles in commercial grain sorghum varieties and suggest the possibility of an unknown cold tolerance regulator which, if identified, could have implications for crop improvement of chilling tolerance outside sorghum.