Mitochondrial Genomes as Tools for Analysis and Teaching of Comparative Genomics, Evolution, and Ecology

Mitochondria produce ATP, the energy molecule necessary for the biosynthetic pathways of life. They are thought to be one of the first landmark symbiotic events; having originated through endosymbiosis of an early alphaproteobacterium into a host cell. The mitochondrial genome has undergone significant changes in organization, size and complexity due to loss of protein coding genes. However, this complexity can vary widely between the major eukaryotic domains of life, with some mitochondria streamlined to less than 1% of the original genome size, particularly in metazoans, while plants and fungi show much larger, less streamlined mitochondrial genomes. Nevertheless, all mitochondrial genomes are small in comparison to the nuclear genome, and are present in high copy number and uniparentally inherited. Embedded in the double membrane of the organelle are five protein complexes that work together to produce ATP. These unique genome features contribute to make the mitochondria a powerful genetic tool for studying broad ecological and evolutionary questions among myriad of species. This thesis interrogates the mitochondrial gene content, exon-intron structure, organization and ancestral states of pertinent genome features for two biologically important species: lichens and diatoms. Lichens are an obligate symbiosis ideal for exploring the genomic consequences of resource sharing involved in a mutualistic relationship. They are estimated to cover up to ten percent of the earth&rsquo;s landmass and contribute importantly to the process of carbon fixation and oxygen production. Diatoms are extremely successful unicellular, photosynthetic, eukaryotic algae that inhabit both freshwater and marine habitats. They have the most efficient RuBisCO enzyme recorded among autotrophs and marine diatoms alone are estimated to perform 20% of the global carbon fixation; an amount comparable to all terrestrial rainforests. These two species have been extensively studied using classical experimental techniques, but have a dearth of genetic resources available. To better understand these extremely important species I harnessed the mitochondrial genome to make comparisons across the lichen and diatom trees of life. In order to examine the genetic content of these mitochondrial genomes, I utilized many different bioinformatics tools. In concert with my own data collection, I helped design a course curriculum, and associated course materials, designed to teach upper level undergraduate and graduate students how to properly assemble and annotated mitochondrial genomes. My thesis not only demonstrates the utility of the mitochondrial genome for answering important evolutionary questions in diverse and important species, it also presents one method for teaching other researchers the tools necessary to assemble and annotate organellar genomes of interest for their own research.</p

Mitochondrial genomes do not appear to regulate flowering pattern/reproductive strategy in Cannabis sativa

Currently, the amount of genetic data for Cannabis is lacking due to the illegal nature of the plant. Our study used 73 Cannabis sativa whole-genome shotgun libraries to reveal eight different mtDNA haplotypes. The most common haplotype contained 60 of the 73 samples studied and was composed of only dioecious individuals. However, other haplotypes contained a mix of both mating strategies (i.e. monoecious and dioecious). From these haplotype groupings we further examined the fully annotated mitochondrial genomes of four hemp individuals with different mt haplotypes and recorded gene content, copy number variation and synteny. Our results revealed highly syntenic mitochondrial genomes that contained ~60 identifiable sequences for protein-coding genes, tRNAs and rRNAs and no obvious rearrangements or chimeric genes. We found no clear evidence that modern reproductive patterns are due to simple cytoplasmic male sterility mutations. It is likely the interaction between nuclear genetic components and the X/Y sex chromosomes that determines reproductive strategy. Additionally, we added 50 % more mitochondrial genomes to the publicly available repository. &nbsp;</p

Patterns of genetic variation in a prairie wildflower, Silphium integrifolium, suggest a non-prairie origin and locally adaptive variation

PREMISE: Understanding the relationship between genetic structure and geography provides information about a species’ history and can be used for breeding and conservation goals. The North American prairie is interesting because of its recent origin and subsequent fragmentation. Silphium integrifolium, an iconic perennial American prairie wildflower, is targeted for domestication, having undergone a few generations of improvement. We present the first application of population genetic data in this species to address the following goals: (1) improve breeding by characterizing genetic structure and (2) identify the species geographic origin and potential targets and drivers of selection during range expansion. METHODS: We developed a reference transcriptome as a genotyping reference for samples from throughout the species range. Population genetic analyses were used to describe patterns of genetic variation, and demographic modeling was used to characterize potential processes that shaped variation. Outlier scans for selection and associations with environmental variables were used to identify loci linked to putative targets and drivers of selection. RESULTS: Genetic variation partitioned samples into three geographic clusters. Patterns of variation and demographic modeling suggest that the species origin is in the American Southeast. Breeding program accessions are from the region with lowest observed genetic variation. CONCLUSIONS: This prairie species did not originate within the prairie. Breeding may be improved by including accessions from outside of the germplasm founding region. The geographic structuring and the identified targets and drivers of adaptation can guide collecting efforts toward populations with beneficial agronomic traits

Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants

Diatoms are the most diverse lineage of algae, but the diversity of their chloroplast genomes, particularly within a genus, has not been well documented. Herein, we present three chloroplast genomes from the genus Halamphora (H. americana, H. calidilacuna, and H. coffeaeformis), the first pennate diatom genus to be represented by more than one species. Halamphora chloroplast genomes ranged in size from ~120 to 150 kb, representing a 24% size difference within the genus. Differences in genome size were due to changes in the length of the inverted repeat region, length of intergenic regions, and the variable presence of ORFs that appear to encode as-yet-undescribed proteins. All three species shared a set of 161 core features but differed in the presence of two genes, serC and tyrC of foreign and unknown origin, respectively. A comparison of these data to three previously published chloroplast genomes in the non-pennate genus Cyclotella (Thalassiosirales) revealed that Halamphora has undergone extensive chloroplast genome rearrangement compared to other genera, as well as containing variation within the genus. Finally, a comparison of Halamphora chloroplast genomes to those of land plants indicates diatom chloroplast genomes within this genus may be evolving at least ~4&ndash;7 times faster than those of land plants. Studies such as these provide deeper insights into diatom chloroplast evolution and important genetic resources for future analyses.</p

Genome streamlining via complete loss of introns has occurred multiple times in lichenized fungal mitochondria.

Reductions in genome size and complexity are a hallmark of obligate symbioses. The mitochondrial genome displays clear examples of these reductions, with the ancestral alpha-proteobacterial genome size and gene number having been reduced by orders of magnitude in most descendent modern mitochondrial genomes. Here, we examine patterns of mitochondrial evolution specifically looking at intron size, number, and position across 58 species from 21 genera of lichenized Ascomycete fungi, representing a broad range of fungal diversity and niches. Our results show that th

Whole Genome Shotgun Sequencing Detects Greater Lichen Fungal Diversity Than Amplicon-Based Methods in Environmental Samples

In this study we demonstrate the utility of whole genome shotgun (WGS) metagenomics in study organisms with small genomes to improve upon amplicon-based estimates of biodiversity and microbial diversity in environmental samples for the purpose of understanding ecological and evolutionary processes. We generated a database of full-length and near-full-length ribosomal DNA sequence complexes from 273 lichenized fungal species and used this database to facilitate fungal species identification in the southern Appalachian Mountains using low coverage WGS at higher resolution and without the biases of amplicon-based approaches. Using this new database and methods herein developed, we detected between 2.8 and 11 times as many species from lichen fungal propagules by aligning reads from WGS-sequenced environmental samples compared to a traditional amplicon-based approach. We then conducted complete taxonomic diversity inventories of the lichens in each one-hectare plot to assess overlap between standing taxonomic diversity and diversity detected based on propagules present in environmental samples (i.e., the &ldquo;potential&rdquo; of diversity). From the environmental samples, we detected 94 species not observed in organism-level sampling in these ecosystems with high confidence using both WGS and amplicon-based methods. This study highlights the utility of WGS sequence-based approaches in detecting hidden species diversity and demonstrates that amplicon-based methods likely miss important components of fungal diversity. We suggest that the adoption of this method will not only improve understanding of biotic constraints on the distributions of biodiversity but will also help to inform important environmental policy.</p

Extensive chloroplast genome rearrangement amongst three closely related Halamphora spp. (Bacillariophyceae), and evidence for rapid evolution as compared to land plants.

Diatoms are the most diverse lineage of algae, but the diversity of their chloroplast genomes, particularly within a genus, has not been well documented. Herein, we present three chloroplast genomes from the genus Halamphora (H. americana, H. calidilacuna, and H. coffeaeformis), the first pennate diatom genus to be represented by more than one species. Halamphora chloroplast genomes ranged in size from ~120 to 150 kb, representing a 24% size difference within the genus. Differences in genome size were due to changes in the length of the inverted repeat region, length of intergenic regions, and the variable presence of ORFs that appear to encode as-yet-undescribed proteins. All three species shared a set of 161 core features but differed in the presence of two genes, serC and tyrC of foreign and unknown origin, respectively. A comparison of these data to three previously published chloroplast genomes in the non-pennate genus Cyclotella (Thalassiosirales) revealed that Halamphora has undergone extensive chloroplast genome rearrangement compared to other genera, as well as containing variation within the genus. Finally, a comparison of Halamphora chloroplast genomes to those of land plants indicates diatom chloroplast genomes within this genus may be evolving at least ~4-7 times faster than those of land plants. Studies such as these provide deeper insights into diatom chloroplast evolution and important genetic resources for future analyses

Genetic Architecture of Capitate Glandular Trichome Density in Florets of Domesticated Sunflower (Helianthus annuus L.)

Capitate glandular trichomes (CGT), one type of glandular trichomes, are most common in Asteraceae species. CGT can produce various secondary metabolites such as sesquiterpene lactones (STLs) and provide durable resistance to insect pests. In sunflower, CGT-based host resistance is effective to combat the specialist pest, sunflower moth. However, the genetic basis of CGT density is not well understood in sunflower. In this study, we identified two major QTL controlling CGT density in sunflower florets by using a F4 mapping population derived from the cross HA 300 × RHA 464 with a genetic linkage map constructed from genotyping-by-sequencing data and composed of 2121 SNP markers. One major QTL is located on chromosome 5, which explained 11.61% of the observed phenotypic variation, and the second QTL is located on chromosome 6, which explained 14.06% of the observed phenotypic variation. The QTL effects and the association between CGT density and QTL support interval were confirmed in a validation population which included 39 sunflower inbred lines with diverse genetic backgrounds. We also identified two strong candidate genes in the QTL support intervals, and the functions of their orthologs in other plant species suggested their potential roles in regulating capitate glandular trichome density in sunflower. Our results provide valuable information to sunflower breeding community for developing host resistance to sunflower insect pests