Homologous recombination represents a DNA repair pathway. Its role in a plant cell is not limited to double strand break repair. It also extends to genome evolution via rearranging of DNA sequences, and has an important application in foreign DNA integration in the plant genome. Our study demonstrated that effects exerted by stress on homologous recombination and genome stability are not restricted to the exposed generation. The progeny of plants exposed to stress exhibited elevated spontaneous homologous recombination, changes in DNA methylation and higher tolerance to stress. These heritable changes are mediated by an unknown stress-inducible epigenetic signal. Furthermore, we demonstrated that using factors that enhance homologous recombination can improve the efficiency of genetic transformation by Agrobacterium. We have developed and patented a plant growth medium enhancing homologous recombination and significantly increasing the transformation frequency. The role of several other chemicals for the improvement of transformation was also evaluated. / xxi, 246 leaves : ill. ; 29 cm. --

Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.

Indiana University-Purdue University Indianapolis (IUPUI) / The black blow fly, Phormia regina (Diptera: Calliphoridae), is a widely abundant fly autochthonous to North America. Like many other Calliphorids, P. regina plays a key role in several disciplines particularly in estimating post-mortem intervals (PMI). The aim of this work was to better understand the population genetic structure of this important ecological species using microsatellites from populations collected in the U.S. during 2008 and 2013. Additionally, it sought to determine the effect of limited genetic diversity on a quantitative trait throughout immature development; larval length, a measurement used to estimate specimen age. Observed heterozygosity was lower than expected at five of the six loci and ranged from 0.529-0.880 compared to expected heterozygosity that ranged from 0.512-0.980, this is indicative of either inbreeding or the presence of null alleles. Kinship coefficients indicate that individuals within each sample are not strongly related to one another; values for the wild-caught populations ranged from 0.033-0.171 and a high proportion of the genetic variation (30%) can be found among samples within regions. The population structure of this species does not correlate well to geography; populations are different to one another resulting from a lack of gene flow irrespective of geographic distance, thus inferring temporal distance plays a greater role on the genetic variation of P. regina. Among colonized samples, flies lost much of their genetic diversity, ≥67% of alleles per locus were lost, and population samples became increasingly more related; kinship coefficient values increased from 0.036 for the wild-caught individuals to 0.261 among the F10 specimens. Colonized larvae also became shorter in length following repeated inbreeding events, with the longest recorded specimen in F1 18.75 mm in length while the longest larva measured in F11 was 1.5 mm shorter at 17.25 mm. This could have major implications in forensic entomology, as the largest specimen is often assumed to be the oldest on the corpse and is subsequently used to estimate a postmortem interval. The reduction in length ultimately resulted in a greater proportion of individuals of a similar length; the range of data became reduced. Consequently, the major reduction in genetic diversity indicates that the loss in the spread of length distributions of the larvae may have a genetic influence or control. Therefore, this data highlights the importance when undertaking either genetic or development studies, particularly of blow flies such as Phormia regina, that collections of specimens and populations take place not only from more than one geographic location, but more importantly from more than one temporal event.

Root rot diseases of bean (Phaseolus vulgaris L.) are a problem wherever they are grown, and are a major constraint to dry edible and snap bean production. Root rot is a primary yield limitation of snap bean production in the US, especially within the top three snap bean producing states of Wisconsin, Oregon and New York. Bean root rot pathogens will be present by the end of the first season even when starting with clean ground. The decline in yield can be relatively slow, so growers might not notice or appreciate the hidden yield cost associated with root rot disease. Traditional methods for disease control such as fungicides, crop rotations, cover crops, seedbed preparations have been proven ineffective (either physically ineffective or economically unviable) against root rot. Therefore, genetic resistance is needed. In order to address the need for genetic resistance to root rot in snap beans, the highly root rot resistant line RR6950, a small seeded black indeterminate type IIIA accession of unknown origin, was crossed with OSU5446, a highly root rot susceptible determinate type I blue lake four-sieve breeding line to produce the RR138 recombinant inbred mapping population. In this study we evaluated the RR138 RI population in the F₆ generation for resistance to Fusarium solani root rot in Oregon and Aphanomyces euteiches root rot in Wisconsin. We also evaluated this population for morphological traits and root structural traits including pod height, pod width, pod length, pod wall thickness, strings, seed color, flower color, tap and basal root diameter, and root angle measurements. The RR138 population was also genotyped on the 10K BeanCAP Illumina Beadchip. The Single Nucleotide Polymorphism (SNP) data was used to assemble a high-density linkage map and Quantitative Trait Loci (QTL) for phenotypic data were evaluated. The linkage map produced from this study contained 1,689 SNPs across 1,196cM. The map was populated with 1 SNP for every 1.4cM, spanning across 11 linkage groups. Three QTL associated with A. euteiches root rot resistance were consistently expressed in 2011 and 2012 trials. A. euteiches QTL were found on Pv02, Pv04, and Pv06 and accounted for 7-17% of total genetic variation. Two QTL associated with F. solani were found in 2011 trial on Pv03 and Pv07, account for 9 and 22% of total genetic variation, respectively. We also found several QTL for morphological traits and root structural traits including QTL for pod fiber and pod height on Pv04, pod length on Pv01, strings on Pv01, taproot diameter on Pv05, and shallow basal root angle on Pv05, accounting for 21, 26, 12, 20, 11, and 19% of total genetic variation, respectively. QTL discovered from Oregon data for F. solani resistance did not cluster with QTL for A. euteiches root rot resistance. "SNP0928_7", was highly associated with F. solani resistance on Pv07 and "SNP0508_2", was highly associated with A. euteiches on Pv02. QTL and markers associated with QTL from this study will be of value to snap bean breeders developing root rot resistant lines with processing traits, and provide more information about targeting the mechanism of resistance. / Graduation date: 2013

Indiana University-Purdue University Indianapolis (IUPUI) / Phormia regina (Meigen), commonly known as the black blow fly is a dipteran that belongs to the family Calliphoridae. Calliphorids play an important role in various research fields including ecology, medical studies, veterinary and forensic sciences. P. regina, a non-model organism, is one of the most common forensically relevant insects in North America and is typically used to assist in estimating postmortem intervals (PMI). To better understand the roles P. regina plays in the numerous research fields, we re-constructed its genome using next generation sequencing technologies. The focus was on generating a reference genome through de novo assembly of high-throughput short read sequences. Following assembly, genetic markers were identified in the form of microsatellites and single nucleotide polymorphisms (SNPs) to aid in future population genetic surveys of P. regina. A total 530 million 100 bp paired-end reads were obtained from five pooled male and female P. regina flies using the Illumina HiSeq2000 sequencing platform. A 524 Mbp draft genome was assembled using both sexes with 11,037 predicted genes. The draft reference genome assembled from this study provides an important resource for investigating the genetic diversity that exists between and among blow fly species; and empowers the understanding of their genetic basis in terms of adaptations, population structure and evolution. The genomic tools will facilitate the analysis of genome-wide studies using modern genomic techniques to boost a refined understanding of the evolutionary processes underlying genomic evolution between blow flies and other insect species.

The United States Pacific Northwest is well known for its shellfish farming. Historically, commercial harvests were dominated by the native Olympia oyster, Ostrea lurida, but over-exploitation, habitat degradation, and competition and predation by non-native species has drastically depleted their densities and extirpated many local populations. As a result, shellfish aquaculture production has shifted to the introduced Pacific oyster, Crassostrea gigas. An underlying objective of this dissertation is the use of molecular genetics to improve our ability to accurately identifying optimal oyster broodstock for either restoration of Olympia oysters or farming of Pacific oysters. The ecological benefits provided by oysters as well as the Olympia oyster's historical significance, has motivated numerous restoration/supplementation efforts but these efforts are proceeding without a clear understanding of the genetic structure among extant populations, which could be substantial as a consequence of limited dispersal, local adaptation and/or anthropogenic impacts. To facilitate this understanding, we isolated and characterized 19 polymorphic microsatellites and used 8 of these to study the genetic structure of 2,712 individuals collected from 25 remnant Olympia oyster populations between the northern tip of Vancouver Island BC and Elkhorn Slough CA. Gene flow among geographically separated extant Olympia oyster populations is surprisingly limited for a marine invertebrate species whose free-swimming larvae are capable of planktonic dispersal as long as favorable water conditions exist. We found a significant correlation between geographic and genetic distances supporting the premise that coastal populations are isolated by distance. Genetic structure among remnant populations was not limited to broad geographic regions but was also present at sub-regional scales in both Puget Sound WA and San Francisco Bay CA. Until it can be determined whether genetically differentiated O. lurida populations are locally adapted, restoration projects and resource managers should be cautious of random mixing or transplantation of stocks where gene flow is restricted. As we transition from our Olympia oyster population analysis to our Pacific oyster quantitative analysis, we recognize that traditional quantitative trait locus (QTL) mapping strategies use crosses among inbred lines to create segregating populations. Unfortunately, even low levels of inbreeding in the Pacific oyster (Crassostrea gigas) can substantially depress economically important quantitative traits such as yield and survival, potentially complicating subsequent QTL analyses. To circumvent this problem, we constructed an integrated linkage map for Pacific oysters, consisting of 65 microsatellite (18 of which were previously unmapped) and 212 AFLP markers using a full-sib cross between phenotypically differentiated outbred families. We identified 10 linkage groups (LG1-LG10) spanning 710.48 cM, with an average genomic coverage of 91.39% and an average distance between markers of 2.62 cM. Average marker saturation was 27.7 per linkage group, ranging between 19 (LG9) and 36 markers (LG3). Using this map we identified 12 quantitative trait loci (QTLs) and 5 potential QTLs in the F1 outcross population of 236 full-sib Pacific oysters for four growth-related morphometric measures, including individual wet live weight, shell length, shell width and shell depth measured at four post-fertilization time points: plant-out (average age of 140 days), first year interim (average age of 358 days), second year interim (average age of 644 days) and harvest (average age of 950 days). Mapped QTLs and potential QTLs accounted for an average of 11.2% of the total phenotypic variation and ranged between 2.1 and 33.1%. Although QTL or potential QTL were mapped to all Pacific oyster linkage groups with the exception of LG2, LG8 and LG9, three groups (LG4, LG10 and LG5) were associated with three or more QTL or potential QTL. We conclude that alleles accounting for a significant proportion of the total phenotypic variation for morphometric measures that influence harvest yield remain segregating within the broodstock of West Coast Pacific oyster selective breeding programs. / Graduation date: 2012