Browsing by Author "Hilu, Khidir W."

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    The acclimation ability of the shale barren endemic Eriogonum alleni to light and heat
    Braunschweig, Suzanne Hill (Virginia Tech, 1993-09-04)
    Shale barrens are unique habitats located throughout the southern Appalachians. They are characterized by a south or south west aspect, a steep slope, and an exposed rocky surface (Platt, 1951). They have a high total irradiance and can experience temperatures higher than the surrounding deciduous forest. A variety of plant species, several of which are rare or endangered, are endemic to the shale barren habitat. One reason proposed fc)r their endemism is that the plants are obligate heliophytes (Keener, 1983). The purpose of this dissertation is tel examine the acclimation ability of the shale barren endemic Eriogonum alIeni to shade and high temperature.
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    Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae
    Barthet, Michelle M.; Moukarzel, Keenan; Smith, Kayla N.; Patel, Jaimin; Hilu, Khidir W. (2015-09-29)
    Background The plastid maturase MatK has been implicated as a possible model for the evolutionary “missing link” between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution. Results Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history. Conclusion These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.
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    Biochemical and Functional Characterization of Induced Terpene Formation in Arabidopsis Roots
    Sohrabi, Reza (Virginia Tech, 2013-08-13)
    Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis.
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    Comparative Genome Analysis of Three Brucella spp. and a Data Model for Automated Multiple Genome Comparison
    Sturgill, David Matthew (Virginia Tech, 2003-07-30)
    Comparative analysis of multiple genomes presents many challenges ranging from management of information about thousands of local similarities to definition of features by combination of evidence from multiple analyses and experiments. This research represents the development stage of a database-backed pipeline for comparative analysis of multiple genomes. The genomes of three recently sequenced species of Brucella were compared and a superset of known and hypothetical coding sequences was identified to be used in design of a discriminatory genomic cDNA array for comparative functional genomics experiments. Comparisons were made of coding regions from the public, annotated sequence of B. melitensis (GenBank) to the annotated sequence of B. suis (TIGR) and to the newly-sequenced B. abortus (personal communication, S. Halling, National Animal Disease Center, USDA). A systematic approach to analysis of multiple genome sequences is described including a data model for storage of defined features is presented along with necessary descriptive information such as input parameters and scores from the methods used to define features. A collection of adjacency relationships between features is also stored, creating a unified database that can be mined for patterns of features which repeat among or within genomes. The biological utility of the data model was demonstrated by a detailed analysis of the multiple genome comparison used to create the sample data set. This examination of genetic differences between three Brucella species with different virulence patterns and host preferences enabled investigation of the genomic basis of virulence. In the B. suis genome, seventy-one differentiating genes were found, including a contiguous 17.6 kb region unique to the species. Although only one unique species-specific gene was identified in the B. melitensis genome and none in the B. abortus genome, seventy-nine differentiating genes were found to be present in only two of the three Brucella species. These differentiating features may be significant in explaining differences in virulence or host specificity. RT-PCR analysis was performed to determine whether these genes are transcribed in vitro. Detailed comparisons were performed on a putative B. suis pathogenicity island (PAI). An overview of these genomic differences and discussion of their significance in the context of host preference and virulence is presented.
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    Contemporaneous radiations of fungi and plants linked to symbiosis
    Lutzoni, Francois; Nowak, Michael D.; Alfaro, Michael E.; Reeb, Valerie; Miadlikowska, Jolanta; Krug, Michael; Arnold, A. Elizabeth; Lewis, Louise A.; Swofford, David L.; Hibbett, David; Hilu, Khidir W.; James, Timothy Y.; Quandt, Dietmar; Magallon, Susana (2018-12-21)
    Interactions between fungi and plants, including parasitism, mutualism, and saprotrophy, have been invoked as key to their respective macroevolutionary success. Here we evaluate the origins of plant-fungal symbioses and saprotrophy using a time-calibrated phylogenetic framework that reveals linked and drastic shifts in diversification rates of each kingdom. Fungal colonization of land was associated with at least two origins of terrestrial green algae and preceded embryophytes (as evidenced by losses of fungal flagellum, ca. 720 Ma), likely facilitating terrestriality through endomycorrhizal and possibly endophytic symbioses. The largest radiation of fungi (Leotiomyceta), the origin of arbuscular mycorrhizae, and the diversification of extant embryophytes occurred ca. 480 Ma. This was followed by the origin of extant lichens. Saprotrophic mushrooms diversified in the Late Paleozoic as forests of seed plants started to dominate the landscape. The subsequent diversification and explosive radiation of Agaricomycetes, and eventually of ectomycorrhizal mushrooms, were associated with the evolution of Pinaceae in the Mesozoic, and establishment of angiosperm-dominated biomes in the Cretaceous.
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    Derivation, fertility and breeding value of doubled monoploids from the diploid potato species, Solanum phureja
    M'Ribu, H. Kabura (Virginia Tech, 1990-08-20)
    Thirty-two monoploids (2n = x = 12) derived by anther culture of ten diploid clones of Solanum phureja were used to generate doubled monoploids through in vitro shoot regeneration. Doubled monoploids were compared to the anther donor and progenitor monoploids for morphological characteristics, and were evaluated for fertility in the greenhouse and progeny performance under field conditions. Monoploids varied for frequency and earliness of shoot regeneration, number of shoots formed per explant and frequency of chromosome doubling among regenerated shoots. Regeneration was greater when stock plantlets were frequently subcultured (2- or 4-week intervals) and maintained under a 16 h photoperiod, and when explants were incubated at 20°C compared to 25°C. In addition, leaf explants regenerated at higher frequencies than stem explants. Significant high correlations between monoploids and their doubled monoploids were observed for 14 of 17 characters in the greenhouse. Doubled monoploids were significantly greater than monoploids for 15 characters, indicating a positive effect of increasing gene dosage from monoploid to diploid. The anther donor was not significantly greater than the mean of doubled monoploids for 10 characters; therefore, for specific characters, doubled monoploids without homozygote depression can be obtained. Doubled monoploids varied for nurnber of days to flower, duration of flowering, abundance of flowers, flower quality, fruit set and seed set; they had lower fruit and seed set than the anther donor. A few clones produced low levels of stainable pollen which had high 2n pollen frequency but did not germinate in vitro. Therefore, they were considered male-sterile for practical purposes. Used as female parents, doubled monoploids were able to transmit the 2n pollen trait to their progenies. Two of four doubled monoploids exhibited superior general combining ability over the anther donor under field conditions. This demonstrates the potential of passage of a heterozygous genotype through the monoploid sieve. The advantage of the monoploid sieve may be more or less evident depending on the combining ability of the crossing partner and variable performance can be expected among doubled monoploids from an unselected anther donor. The performance of unselected doubled monoploids demonstrates the potential for their utilization in breeding and warrants further research in the area.
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    Dissection of Drought Responses in Arabidopsis
    Harb, Amal Mohammad (Virginia Tech, 2010-07-19)
    Plants as sessile organisms are susceptible to many environmental stresses such as drought, and salinity. They have therefore evolved mechanisms to acclimate and tolerate environmental stresses. Knowledge of the molecular aspects of abiotic stress gleaned from extensive studies in Arabidopsis has provided much information on the complex processes underlying plant response to abiotic stresses. Nevertheless, there is a need for integration of the knowledge gained and a systematic molecular genetic dissection of the complex responses to abiotic stress. In this study in Arabidopsis, comparative expression profiling analysis of progressive (pDr) and moderate (mDr) drought treatments revealed common drought responses, as well as treatment specific signatures responses to drought stress. Under prolonged moderate drought plants develop different mechanisms for acclimation: induction of cell wall loosening at early stage, and a change in hormonal balance (ABA: JA) at late stage of moderate drought. Taking a reverse genetics approach, a MYB transcription factor (MYB109) has been identified as a regulator of growth under drought and salt stress. Global expression profiling showed possible mechanisms of how MYB109 modulates growth under drought conditions: as a regulator of RNA processing and splicing and as a negative regulator of jasmonic acid biosynthesis and signaling. A forward genetics screen for drought and salt tolerance of transposon activation tag (ATag) lines led to the discovery of novel genes, which shed light on unexplored areas of abiotic stress biology. Utilizing this strategy, a potential role for cell wall modification and MATE transporters in response to drought and salt stress has been discovered, which needs further analysis to integrate this information on the role of these biological processes in plant stress biology.
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    Effect of Capsaicin Supplementation on Performance of and Physiological Response to Repeated Sprinting
    Opheim, Maximilian Nicholas (Virginia Tech, 2010-02-02)
    Aim: Fatigue during team sports requiring multiple sprints can result from the combined effects of metabolic, mechanical, neurological, and immune factors. The purpose of this study was to investigate the influence of capsaicin on performance of and the physiological response to an exercise test simulating the fitness demands of team sport game conditions. Methods: This study was a placebo-controlled, crossover design. Nineteen healthy male experienced athletes age 18-30 yr consumed either 3 g/d cayenne (25.8 mg/d capsaicin) or placebo for 1 wk. Directly following the supplementation period, they completed a repeated sprint test consisting of 15 30 m maximal effort sprints on 35 s intervals. Sprint times were recorded via electronic dual-beam timing system. Fasted blood draws for interleukin-6 (IL-6) were taken at baseline prior to supplementation, 45-min pretest, and immediately post test. Heart rate (HR), blood pressure (BP), rate of perceived exertion (RPE), muscle soreness (MS), and gastrointestinal distress (GD) were measured 1-min pretest, during, posttest, and 1-min posttest. MS was also measured for 3 d posttest. Results: Relative to the placebo, capsaicin significantly reduced maximum HR by 9.3%, total average HR by 8.5%, and sprinting average HR by 6.0% (P<0.05). Capsaicin caused GD of at least 2/5 in 24.5% of subjects. There was no difference between treatments in fastest or mean sprint time, fatigue, percent change or difference in IL-6, BP, RPE, sprint or posttest MS. Conclusion: Capsaicin did not influence repeated sprint performance or the inflammatory response, but reduced HR during intense activity and causes substantial GD.
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    Evolutionary history of the canary grasses (Phalaris, Poaceae)
    Voshell, Stephanie (Virginia Tech, 2014-06-12)
    Canary grasses (Phalaris, Poaceae) include 21 species widely distributed throughout temperate and subtropical regions of the world with centers of diversity in the Mediterranean Basin and western North America. The genus contains annual/perennial, endemic/cosmopolitan, wild, and invasive species with basic numbers of x=6 (diploid) and x=7 (diploid/tetraploid/hexaploid). The latter display vastly greater speciation and geographic distribution. These attributes make Phalaris an ideal platform to study species diversification, dispersal, historic hybridization, polyploidy events, and chromosome evolution in the grasses. This body of research presents the first molecular phylogenetic and phylogeographic reconstruction of the genus based on the nuclear ITS and plastid trnT-F DNA regions allowing species relationships and the importance of polyploidy in speciation to be assessed. Divergence dates for the genus were determined using Bayesian methods (BEAST, version 1.6.2) and historic patterns of dispersal were analyzed with RASP (version 2.1b). Self-incompatibility and the feasibility of hybridization between major groups within the genus were studied with a series of greenhouse experiments. Acetocarmine and fluorescent staining techniques were used to study the morphology of the chromosomes in a phylogenetic context and the nuclear DNA content (C values) was quantified using flow cytometry. Four major clades were revealed in the genus with cytological and geographic affinities leading to the establishment of two subgenera and four sections in the first comprehensive infrageneric treatment of Phalaris. Divergence dating revealed a Miocene emergence (20.6-8.4 MYA) for the genus which is concurrent with studies of other genera in the Aveneae tribe. The hypothesis stating that Phalaris originated in the Mediterranean Basin and dispersed to the New World via a western route leading to a secondary center of diversification in western North America was supported by phylogeographic and cytological analyses. An empirical study comparing the weight, length, and width of the florets by morphological type and cytotype revealed significant differences that support a dispersal advantage among the New World and Arundinacea species. The x=6 species displayed greater intraspecific C value variation, higher DNA content per haploid chromosome set, and a distinct karyotype compared with the x=7 species indicating a complex history of chromosome evolution.
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    Evolutionary relationships in Oryza inferred from the gene that encodes the 10 kDa prolamin (seed storage protein) polypeptide
    Mullins, Irene M. (Virginia Tech, 1997-12-01)
    Cereals represent one of the most important food crops in the world. Rice (Oryza sativa) is one of the most commonly consumed cereal grains, and as a result, has considerable economic and agricultural importance, despite their potential as a source of genetic material. The phylogenetic relationships among rice and its wild species are not well understood. The objectives of this study are to evaluate the rate and type of molecular variation present in the gene that encodes the 10 kDa prolamin polypeptide in Oryza (Poaceae), and use this information in understanding the evolution of the Oryza genus. Our principle hypothesis is that genetic diversity exists at the molecular level in wild species of Oryza, and that this diversity can provide useful information regarding the phylogenetic relationships among rice species and permit a more theoretical examination of the evolutionary processes, such as concerted evolution, within Oryza and its nine genomes. A phylogeny of Oryza is presented, and modes of evolution are discussed.
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    Expression and Function of the Chloroplast-encoded Gene matK
    Barthet, Michelle Marie (Virginia Tech, 2006-02-09)
    The chloroplast matK gene has been identified as a rapidly evolving gene at nucleotide and corresponding amino acid levels. The high number of nucleotide substitutions and length mutations in matK has provided a strong phylogenetic signal for resolving plant phylogenies at various taxonomic levels. However, these same features have raised questions as to whether matK produces a functional protein product. matK is the only proposed chloroplast-encoded group II intron maturase. There are 15 genes in the chloroplast that would require a maturase for RNA splicing. Six of these genes have introns that are not excised by a nuclear imported maturase, leaving MatK as the only candidate for processing introns in these genes. Very little research has been conducted concerning the expression and function of this important gene and its protein product. It has become crucial to understand matK expression in light of its significance in RNA processing and plant systematics. In this study, we examined the expression, function and evolution of MatK using a combination of molecular and genetic methods. Our findings indicate that matK RNA and protein is expressed in a variety of plant species, and expression of MatK protein is regulated by development. In addition, matK RNA levels are affected by light. Furthermore, genetic analysis has revealed that although MatK has a high rate of amino acid substitution, these substitutions are not random but are constrained to maintain overall chemical structure and stability in this protein. We have also identified an alternate start codon for matK in some plant species that buffers indels (insertions and deletions) in the open reading frame (ORF) that are not in multiples of three in the gene sequence. Usually, indels not in multiples of three result in frame shifts that destroy the reading frame. Our results indicate that an out-of-frame matK start codon in some orchids compensates for these otherwise deleterious indels. This research represents the first in-depth analysis of matK gene expression and contributes to several fields of biology including plant systematics, genetics and gene expression.
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    Generic concepts in the Crepidotaceae as inferred from nuclear large subunit ribosomal DNA sequences, morphology, and basidiospore dormancy patterns
    Aime, Mary Catherine (Virginia Tech, 1998-11-19)
    The Crepidotaceae (Imai) Singer (Basidiomycetes: Agaricales) represents a proposed family of saprophytic fungi containing five agaricoid (Crepidotus, Tubaria, Melanomphalia, Simocybe, Pleurotellus) and four cyphelloid (Episphaeria, Phaeosolenia, Pellidiscus, Chromocyphella) genera. Several contemporary classification systems exist that delegate some or all of these genera to other agaric families. Phylogenetic relationships for the most prevalent genera in the Crepidotaceae were investigated using nuclear large subunit ribosomal DNA (LSU rDNA) sequences. Parsimony analysis of the molecular data supports the Singer classification of Crepidotus, Melanomphalia, and Simocybe as a single monophyletic unit within the Agaricales. The affinities of the genus Tubaria remain uncertain. Crepidotus (Fr.) Staude is the largest and most phenotypically variable genus in the Crepidotaceae. Sequencing of the LSU rDNA region for a cross-section of morphologically diverse species suggests that Crepidotus is not a monophyletic genus. Analysis of morphological characters for 23 Crepidotus taxa shows that characters traditionally applied for infrageneric classification of Crepidotus are homoplasic in origin, but that less commonly emphasized characters such as spore shape and ultrastructure of spore wall ornamentation may be indicative of monophyletic clades for this complex. A unique pattern of basidiospore dormancy and germination, unknown in any other species of agaric, is reported for 11 species of Crepidotus. Similar patterns were also encountered in species of Simocybe and Melanomphalia. In these species an endogenous period of spore dormancy of four to six months is followed by an activation period where the factors necessary for subsequent germination appear to involve a minimal nutritional component, water, and exposure to light.
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    Genetical and molecular systematic study on the genus Montagnea Fr., a desert adapted Gasteromycete
    Chen, Chang (Virginia Tech, 2004-04-30)
    Montagnea arenaria [Hymenogastrales, Basidiomycota] , adapted to desert and xeric habitats, is morphologically and phenotypically variable. Species have been described on the basis of macromorphology and spore shape and size. This study was initiated to investigate populations of M. arenaria from Namibia in Africa and the Southwestern United States. It was hypothesized that biological species would exist in the widely separated populations. Spores from single sporocarps were germinated, single spore isolates were obtained and selfed to obtain mating types. On transfer, clamp connections were not maintained and mating patterns could not be achieved. Nuclear staining revealed multinuclei in the hyphae of both single spore isolates and compatible crosses. Spores were stained and found to have either 1 or 2 nuclei, but only four sterigmate basidia were observed. Limited partial compatibility was achieved and in some cases clamp connections formed within and between crosses from the two continents. Genomic DNA was extracted from old herbarium specimens. The ITS1, 5.8S, and ITS4 regions of nuclear ribosomal DNA were amplified and sequenced directly. Phylogenetic analysis using PAUP was performed. The hypothesis that Montagnea would form different biological species based on continental separation was rejected. In fact, the complex of isolates from widely varying locations not only had partial compatibility, but the variation in ITS sequences among widely distributed collections was relatively low. Lastly, no correlation between sporocarp size and gene flow among specimens from a wide variety of habitats was found. It appears that M. arenaria is a single, highly variable, widely distributed species.
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    Impact of Gene Molecular Evolution on Phylogenetic Reconstruction: A Case Study in the Rosids (Superorder Rosanae, Angiosperms)
    Hilu, Khidir W.; Black, Chelsea M.; Oza, Dipan (PLOS, 2014-06-16)
    Rate of substitution of genomic regions is among the most debated intrinsic features that impact phylogenetic informativeness. However, this variable is also coupled with rates of nonsynonymous substitutions that underscore the nature and degree of selection on the selected genes. To empirically address these variables, we constructed four completely overlapping data sets of plastid matK, atpB, rbcL, and mitochondrial matR genes and used the rosid lineage (angiosperms) as a working platform. The genes differ in combinations of overall rates of nucleotide and amino acid substitutions. Tree robustness, homoplasy, accuracy in contrast to a reference tree, and phylogenetic informativeness are evaluated. The rapidly evolving/unconstrained matK faired best, whereas remaining genes varied in degrees of contribution to rosid phylogenetics across the lineage's 108 million years evolutionary history. Phylogenetic accuracy was low with the slowly evolving/unconstrained matR despite least amount of homoplasy. Third codon positions contributed the highest amount of parsimony informative sites, resolution and informativeness, but magnitude varied with gene mode of evolution. These findings are in clear contrast with the views that rapidly evolving regions and the 3rd codon position have inevitable negative impact on phylogenetic reconstruction at deep historic level due to accumulation of multiple hits and subsequent elevation in homoplasy and saturation. Relaxed evolutionary constraint in rapidly evolving genes distributes substitutions across codon positions, an evolutionary mode expected to reduce the frequency of multiple hits. These findings should be tested at deeper evolutionary histories.
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    The Inhibitory Effect of Rhododendron maximum L. (Ericaceae) Thickets on Mycorrhizal Colonization of Canopy Tree Seedlings
    Walker, John F. (Virginia Tech, 1998-04-28)
    Thickets of Rhododendron maximum (Rm) in the southern Appalachians impose severe limitations on the regeneration of hardwood and coniferous seedlings. Interactions between Rm thickets and ectomycorrhizal colonization were examined to explain seedling inhibition. Experimental blocks were established in and out of Rm thickets in a mature, mixed hardwood/conifer forest in Macon County, North Carolina. Planted seedlings of red oak (Quercus rubra) and hemlock (Tsuga canadensis) were harvested at the end of the first and second growing seasons. Litter manipulation had no effect on total mycorrhizal colonization. Mycorrhizal colonization and ramification index (# mycorrhizae cm-1) were depressed and colonization by Cenococcum geophilum increased in blocks with versus without Rm. After the first year, percent colonization of T. canadensis not in Rm thickets (62 %) was three times higher than in Rm thickets (19%), and the ramification index was increased by more than a factor of four (2.83 versus 0.61). Mycorrhizal colonization levels were correlated with root weight and shoot weight in both hemlock and oak seedlings, but did not explain most of the variation observed. Sporocarps of 69 putatively ectomycorrhizal species were collected on the blocks. Species diversity and overall community structure was similar in and out of Rm thickets. Individual species, i.e. Lactarius speciosus and Russula krombholzii, were significant indicators of forest without Rm thickets. Rhododendron maximum thickets probably affect the process of mycorrhization. The reduced level of mycorrhizal capacity under Rm thickets could be a factor in the increased seedling failure in Rm thickets.
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    Karyotype evolution in Phalaris (Poaceae): The role of reductional dysploidy, polyploidy and chromosome alteration in a wide-spread and diverse genus
    Winterfeld, Grit; Becher, Hannes; Voshell, Stephanie; Hilu, Khidir W.; Roeser, Martin (PLOS, 2018-02-20)
    Karyotype characteristics can provide valuable information on genome evolution and speciation, in particular in taxa with varying basic chromosome numbers and ploidy levels. Due to its worldwide distribution, remarkable variability in morphological traits and the fact that ploidy change plays a key role in its evolution, the canary grass genus Phalaris (Poaceae) is an excellent study system to investigate the role of chromosomal changes in species diversification and expansion. Phalaris comprises diploid species with two basic chromosome numbers of x = 6 and 7 as well as polyploids based on x = 7. To identify distinct karyotype structures and to trace chromosome evolution within the genus, we apply fluorescence in situ hybridisation (FISH) of 5S and 45S rDNA probes in four diploid and four tetraploid Phalaris species of both basic numbers. The data agree with a dysploid reduction from x = 7 to x = 6 as the result of reciprocal translocations between three chromosomes of an ancestor with a diploid chromosome complement of 2n = 14. We recognize three different genomes in the genus: (1) the exclusively Mediterranean genome A based on x = 6, (2) the cosmopolitan genome B based on x = 7 and (3) a genome C based on x = 7 and with a distribution in the Mediterranean and the Middle East. Both auto-and allopolyploidy of genomes B and C are suggested for the formation of tetraploids. The chromosomal divergence observed in Phalaris can be explained by the occurrence of dysploidy, the emergence of three different genomes, and the chromosome rearrangements accompanied by karyotype change and polyploidization. Mapping the recognized karyotypes on the existing phylogenetic tree suggests that genomes A and C are restricted to sections Phalaris and Bulbophalaris, respectively, while genome B occurs across all taxa with x = 7.
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    Molecular evolution of genes encoding allergen proteins in the peanuts genus Arachis: Structural and functional implications
    Hilu, Khidir W.; Friend, Sheena A.; Vallanadu, Viruthika; Brown, Anne M.; Hollingsworth, Louis R. IV (PLOS, 2019-11-01)
    Food allergies are severe immune responses to plant and animal products mediated by immunoglobulin E (IgE). Peanuts (Arachis hypogaea L.) are among the top 15 crops that feed the world. However, peanuts is among the “big eight food allergens”, and allergies induced by peanuts are a significant public health problem and a life-threatening concern. Targeted mutation studies in peanuts demonstrate that single residue alterations in these allergen proteins could result in substantial reduction in allergenicity. Knowledge of peanut allergen proteins is confined to the allotetraploid crop and its two progenitors. We explored frequencies and positions of natural mutations in the hyperallergenic homologues Ara h 2 and Ara h 6 in newly generated sequences for 24 Arachis wild species and the crop species, assessed potential mutational impact on allergenicity using immunoblots and structural modeling, and evaluated whether these mutations follow evolutionary trends. We uncovered a wealth of natural mutations, both substitutions and gaps, including the elimination of immunodominant epitopes in some species. These molecular alterations appear to be associated with substantial reductions in allergenicity. The study demonstrated that Ara h 2 and Ara h 6 follow contrasting modes of natural selection and opposing mutational patterns, particularly in epitope regions. Phylogenetic analysis revealed a progressive trend towards immunodominant epitope evolution in Ara h 2. The findings provide valuable insight into the interactions among mutations, protein structure and immune system response, thus presenting a valuable platform for future manipulation of allergens to minimize, treat or eliminate allergenicity. The study strongly encourages exploration of genepools of economically important plants in allergenicity research.
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    Molecular Systematics of the Entomopathogenic Bacteria Bacillus popilliae, Bacillus lentimorbus, and Bacillus sphaericus
    Lampe, Karen Rippere (Virginia Tech, 1998-09-11)
    Bacillus popilliae and B. lentimorbus, causative agents of milky disease in Japanese beetles and related scarab larvae, have been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. Thirty-four isolates of these bacteria were examined for DNA similarity. Three distinct but related similarity groups were identified; the first contained strains of B. popilliae, the second contained strains of B. lentimorbus, and the third contained two strains distinct from but related to B. popilliae. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae." Geographically distinct strains of B. popilliae and B. lentimorbus were analyzed using RAPD. Eight decamer primers were tested against nineteen new and seventeen isolates previously described by randomly amplified polymorphic DNA (RAPD) analysis (M. Tran). Of the new isolates, ten were found to be B. popilliae while nine isolates were more related to the B. lentimorbus species. Paraspore formation, believed to be a characteristic unique to B. popilliae, was found to occur among a subgroup of B. lentimorbus strains. Using a combination of two PCR primer pairs, the cry18Aa1 gene was detected in 31 of 35 B. popilliae isolates and in 1 of 18 B. lentimorbus isolates. When hemolymph smears were examined microscopically, a parasporal crystal was seen in three of the four B. popilliae strains where the PCR primers could not amplify the paraspore gene. The fourth strain was not tested due to the unavailability of infected hemolymph. A paraspore was also detected by microscopic examination in a subgroup of 14 B. lentimorbus strains. In combination, the primer pairs CryBp1 and CryBp2 are effective at detecting the paraspore gene in B. popilliae isolates, but not in the B. lentimorbus isolates. Growth in media supplemented with 2% NaCl was found to be less reliable in distinguishing the species than was vancomycin resistance, the latter present only in B. popilliae. The basis for vancomycin resistance in all isolates was investigated using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci. An amplicon was identified and sequenced. The amplified portion of the putative ligase gene in B. popilliae had 77% and 68-69% nucleotide identity to the sequences of the vanA gene and the vanB genes, respectively. There was 75% and 69-70% identity between the deduced amino acid sequence of the putative ligase gene in B. popilliae and the deduced amino acid sequence of the vanA gene and the vanB genes, respectively. It has been determined that the vanE gene is located either on a plasmid greater than 16 kb in size or on the chromosome. The gene in B. popilliae may have had an ancestral gene in common with vancomycin resistance genes in enterococci. Bacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16S rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity.
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    Morphology, Molecular Phylogeny and Genome content of Bothriochloa focusing on Australian taxa
    Sumadijaya, Alex (Virginia Tech, 2015-06-19)
    The study focuses on the genus Bothriochloa (Andropogoneae, Poaceae) in Australia. Despite morphological features separating this genus from the closely related two genera Capillipedium and Dichanthium, (the three hereafter will be called BCD), De Wet and Harlan introduced the compilospecies complex to show the interbreeding phenomena that occurred among species of these genera. This study was carried out to assess species/genus relatedness of the BCD complex using different evidences from morphology, molecular information and genomic content. Nineteen morphological characters were observed, three regions (trnT-F, rps16 intron and 3'trnK) of chloroplast genome phylogenetic were used in phylogenetic reconstruction, and chromosome counting as well as flow cytometry for chromosome number and genome size were conducted during the study. Phylogenetic trees were constructed using MP with NJ for morphological data, and MP, RAxML, and BI for molecular data. Based on morphology, all three genera were separated as monophyletic units. Bothriochloa consisted of two clades. However, phylogenetic analyses based on chloroplast genomic regions reveal that Bothriochloa and Dichanthium are paraphyletic clades and only Capillipedium is resolved as a monophyletic clade. The concatenated data set has performed better than individual data sets in terms of resolution and support for clades. Flow-cytometry and chromosome counting only found diploid and tetraploid but not hexaplod species. TCS network reveals that tetraploidization followed different pathways from the ancestral diploid species. This study provided new insight onto the evolution of the chloroplast genome in the compilospecies and empirical evidence of species grouping of the compilospecies based on morphology.
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    Nutrient and Antinutrient Content of an Underexploited Malawian Water Tuber Nymphaea petersiana (Nyika)
    Chawanje, Chrissie Maureen (Virginia Tech, 1998-12-02)
    Nymphaea petersiana Klotzsch (Nyika) is an important wild tuber eaten in some districts of Malawi. The tubers were processed by boiling/freeze-drying(BFD) and sun-drying(USD). The tuber's nutrient and antinutrient composition was determined to produce a preliminary nutrient data base for use in sub-Saharan Africa. There was no significant difference (P > 0.05) in protein content of BFDand USD samples. Sun-dried samples were significantly (P < .05) higher in ash than boiled samples while boiled samples were significantly higher (P < .05) in crude fat and total carbohydrate. The protein content of the tubers (8.0 and 8.1 %) was higher than that of the staple maize (7.9%), African millets (unspecified) (7.5%), and polished rice (7.0%), but lower than sorghum (10.7%). Protein content was higher than tubers like cassava (1.3%), potato (2%), sweet potato (1.6%), yams (1.5%) and N. lotus (5.2). Nyika tubers have a well balanced amino acid content, limiting only in lysine. There were no significant (P > 0.05) differences in the mineral content of BFD and USD samples, except for iron, which was lower in the boiled samples. Nyika tubers have a higher calcium (1376 and 946 ug/g) and phosphorus (2250 and 2883 ug/g ) content than wild and domesticated cassava, potatoes, sweet potatoes and wild and domesticated yams. Sun-dried tubers have a higher iron content (88ug/g) than maize (20ug/g). The zinc content of tuber was higher (21and 25ug/g) than that of boiled maize flour, boiled sorghum flour, rice, cassava, and sweet potato. The predominant fatty acids in the tubers were oleic (47%), linoleic (32%), palmitic (21%) and linolenic (7%) acids. Ascorbic acid content was very low, only 0.1 and 0.003 mg/100g. Tannin content was lower (1 and 1.7 %) in the tubers than in Vulgare Pers. sorghum, DeKalb sorghum from U.S. and Kabale sorghum from Uganda. There was a significantly (P < 0.05) lower content of phytate in boiled (3.9ug/g) than in sun-dried tubers (6.0 ug/g). Phytate content of the tubers was lower than that of cooked maize flour, unrefined maize flour, cassava and sweet potato. Trypsin inhibitor activity in the tubers was reduced from 463 to 55 TIU/g tuber and chymotrypsin inhibitor activity was reduced to 50 from 267 CIU/g tuber by cooking. Nyika is a good source of iron and quality protein limiting only in lysine. Protein is comparable to staple maize and higher than root crops consumed in Malawi. It is not a good source of fat and ascorbic acid. Tannin, phytate, trypsin, and chymotrypsin inhibitor content lower than most food crops consumed in Malawi.