Clostridium perfringens é um dos microrganismos mais freqüentemente envolvidos em surtos de enfermidades transmitidas por alimentos. Este microrganismo pode ser classificado em cinco tipos toxigênicos (A-E), de acordo com a detecção dos genes codificadores de suas principais toxinas: alfa (cpa), beta (cpb), épsilon (etx) e iota (iap), sendo que técnicas moleculares empregando a PCR são atualmente utilizadas para genotipagem desses isolados. Alguns isolados de C. perfringens produzem uma enterotoxina (CPE) que é responsável pelos sintomas clínicos desenvolvidos em casos de toxinfecção alimentar, sendo que esta toxina é codificada pelo gene cpe. A simples detecção de C. perfringens em um alimento, mesmo naqueles suspeitos de causar surtos, não é suficiente para considerá-lo como de risco à saúde do consumidor. Isto porque dentre os isolados de C. perfringens apenas um número muito pequeno apresenta o gene cpe. Além disso, isolados de C. perfringens não produtores de CPE estão amplamente disseminados no ambiente, em alimentos e mesmo em fezes de pessoas. Desta forma, com o presente estudo verificou-se a freqüência de C. perfringens dentre isolados de clostrídios sulfito redutores, a freqüência de C. perfringens potencialmente enterotoxigênicos e sua variabilidade genética, de modo a evidenciar a importância dessas cepas como causadoras de doenças, além de fornecer subsídios para melhorar os conhecimentos sobre as características das cepas circulantes em nosso meio. Foram utilizados 335 isolados de clostrídios sulfito redutores provenientes de alimentos (126), solo (84) e fezes de animais (125). Dos 335 isolados, 146 (43,6%) foram caracterizados, através de reações bioquímicas e moleculares, comoC. perfringens, sendo 75 isolados (59,5%) provenientes de alimentos, 43 (51,2%) de solo e 28 (22,4%) de fezes de animais. Todas as cepas de C. perfringens analisadas foram tipadas como C. perfringens tipo A. Dos 75 isolados de C. perfringens provenientes de alimentos, 20 apresentaram o gene cpe, sendo 13 (65%) com localização cromossomal; nas demais cepas não foi possível determinar sua localização. Nos isolados de C. perfringens provenientes de solo e das fezes de animais não se verificou a presença desse gene. Das 20 cepas de C. perfringens que apresentaram o gene cpe detectou-se em 15 a produção de enterotoxina; as cinco cepas restantes não apresentaram esporulação no meio DUNCAN STRONG modificado, não sendo possível avaliar sua atividade enterotoxigênica. As 146 cepas de C. perfringens quando submetidas à PFGE geraram 69 perfis PFGE distintos, sendo 42 exclusivos para uma única cepa, indicando uma grande variabilidade genética, entre isolados provenientes de amostra de alimentos, fezes ou solo. A utilização de clostrídios sulfito redutores, ou mesmo de C. perfringens como indicador de possível risco à saúde dos consumidores pode levar à condenação desnecessária de alimentos, uma vez que existe baixa correlação entre costrídios sulfito redutores e C. perfringens, independente da fonte de isolamento, além da baixa freqüência do gene cpe nas cepas estudadas. / Clostridium perfringens is one of the most frequently microorganism involved in outbreaks of foodborne diseases. This microorganism can be classified into five toxigenic types (A to E), according to the detection of genes encoding its major toxins: alpha (cpa), beta (cpb), epsilon (etx) and iota (iap). Molecular techniques using PCR are currently used for genotyping this isolates. Besides the major toxins, some isolates of C. perfringens produce an enterotoxin (CPE) that is responsible for clinical symptoms developed in cases of food poisoning. This enterotoxin is encoded by the cpe gene. The simple detection of C. perfringens in food, even in those suspected of causing outbreaks, is not enough to consider it as a risk to consumers´ health. This happens because among the isolates ofC. perfringens only a very small number shows the cpe gene. In addition, isolates of C. perfringens that do not produce CPE are widespread in the environment, food and even in feces of humans. Thus, the present study examined the frequency of C. perfringens isolates among sulfite reducing clostridia, the frequency of potentially enterotoxigenic C. perfringens and its genetic variability in order to highlight the importance of these strains in causing diseases, and provides subsidies to improve the knowledge about the strains that are circulating in our environment. A total of 335 isolates of sulfite reducing clostridia from foods (126), soil (84) and animal feces (125) were used. Among the 335 isolates, 146 (43.6%) were characterized by biochemical and molecular reactions as C. perfringens, being 75 (59.5%) from foods, 43 (51.2%) from soil and 28 (22.4%) from animal feces. All strains of C. perfringens were typed as C. perfringens type A. Of the 75 isolates of C. perfringens from food, 20 had the cpe gene, and in 13 (65%) the gene was chromosomally located. In the other strains it was not possible to determine the location of this gene. In isolates of C. perfringens from soil and animal feces the cpe gene was not present. Amongst the 20 strains of C. perfringens positive for cpe, enterotoxin production was detected in 15. Five strains showed no sporulation in the medium modified Duncan Strong, being not possible to verify their enterotoxigenic activity. All C. perfringens were subjected to PFGE and generated 69 different PFGE profiles, being 42 unique to a single strain, indicating a great genetic variability among isolates from food, feces or soil. The use of sulfite reducing clostridia, or even C. perfringens as an indicator of possible health risk to consumers can lead to unnecessary condemnation of food, since there is low correlation between sulfite reducing clostridia and C. perfringens, regardless of source of isolation. This study also shows a low frequency of cpe gene in the strains indicating the low risk in causing foodborne disease.

Resumo: O objetivo do presente trabalho foi estudar a divergência genética entre árvores matrizes de Tabebuia caraiba (Mart.) Bur. no município de Macapá, no Estado do Amapá, por meio de caracteres biométricos de flores, frutos, sementes e processo germinativo. A divergência genética foi avaliada pela análise de agrupamento, através do método de Ward, K-means e pelo algoritmo de Tocher, obtidos a partir da matriz de dissimilaridade pela Distância Euclidiana. Para verificação da importância relativa de cada variável para a divergência genética utilizou-se a análise de Componentes Principais. As 119 árvores matrizes foram distribuídas em 21 grupos no método de Ward e 23 grupos para o algoritmo de Tocher. O método K-means auxiliou na exclusão de 11 caracteres pouco discriminatórios. Dos caracteres mais importantes para a divergência genética, destacam-se o comprimento do fruto, largura do fruto, comprimento da ala maior da semente, massa de matéria seca da semente, largura do cotilédone, largura da folha e massa de matéria seca da plântula. Há variabilidade entre as árvores matrizes de T. caraiba quanto aos caracteres avaliados e o estudo da divergência possibilita a identificação de árvores matrizes para a colheita de sementes, que subsidiem programas de conservação genética / Abstract: The objective of this work was to study the genetic diversity among trees of Tabebuia caraiba (Mart.) Bur. at city of Macapá, Amapá State, by means of biometric characters of flowers, fruits, seeds and germination process. Genetic divergence was assessed by cluster analysis by Ward method, K-means algorithm and the Tocher, obtained from the matrix of the Euclidean Distance. To verify the relative importance of each variable to the genetic divergence we used the Principal Component analysis. The 119 selected trees were divided into 21 groups in the method of Ward and 23 groups for the algorithm Tocher. The K-means method aided in the exclusion of 11 characters less discriminatory. Among the most important traits for genetic divergence, we highlight the fruit length, fruit width, length of greater wing of the seed dry weight of seed, cotyledon width, leaf width and dry weight of seedlings. There is variability among the trees of T. caraiba about the traits and the study of divergence allows the identification of mother trees for seed collection, programs that support conservation genetics / Orientadora: Fabíola Vitti Môro / Coorientador: Rinaldo César de Paula / Banca: Dilermando Perecin / Banca: Fabiano Cesarino / Mestre

Most of what is known about the ways in which strongly interacting species affect ecological communities stems from changes to community structure revealed in contemporary research. However, trophic downgrading has limited the temporal extent to which inferences can be drawn. The aim of my Dissertation was to expand on the strongly interacting species concept by examining species interactions at a historical scale, in a textbook example of a strongly interacting and keystone predator. The sea otter, Enhydra lutris, was driven to near-extinction but is recovering in parts of its range, providing a mosaic of areas with and without sea otters. This mosaic allowed for a series of natural experiments, which I conducted using behavioural observations, genetic tools, and archaeological methods, to examine sea otter effects spanning contemporary (last ~40 yrs.), and late-Holocene (~3500-150 yrs. ago) timeframes, and on an evolutionary scale that inferred middle-Pleistocene interactions. In Chapter 2, my coauthors and I found that sea otter use of clam-based niches increased as occupancy-time increased, and that bachelor groups of male otters primarily inhabited these niches, findings that informed and inspired subsequent questions. In Chapter 3, we found that where sea otters were established for 20-30 years, the disturbance to eelgrass (Zostera marina), caused by sea otters digging for clams and other infaunal prey, was correlated with ~25% greater eelgrass allelic richness than where otters were present <10 yrs, or absent. We posit that sea otter digging has long-influenced the genetic diversity and resilience of eelgrass – perhaps since the middle Pleistocene. In Chapter 4, we asked how two strongly interacting species – people and sea otters – co-existed for millennia where they both consumed clams. We used assemblages of live and otter-cracked butter clams (Saxidomus gigantea), to confirm the ecological effects that sea otters exert today. We measured clams from archaeological assemblages in areas densely populated with clam gardens – terraced beaches that enhance clam habitat and productivity – and found that sea otters reduced the sizes of ancient clams, acting as ecologically effective predators in the mid-to-late Holocene. However, clam harvests were stable for thousands of years, with or without otters. We suggest that clam gardening supported coexistence of people and otters in the past, and could function the same way today. Collectively, we found that a few, perhaps long-forgotten, interactions increased the breadth of the strongly interacting species concept. In Chapter 5, I suggest that such rediscoveries could occur in other systems. Many large vertebrates have suffered population declines, but the most insidious losses accompanying these, are the losses of ecological interactions that become unknowable, and thus cannot be intentionally restored. By searching out ancient interactions, long-forgotten relationships have the potential to be recovered, and to inform our understanding of contemporary systems. / Graduate / 2022-09-10

Thesis (M.Sc. Agriculture (Animal Production)) -- University of Limpopo, 2020 / The smallholder dairy sector in South Africa is characterized by a low input production system and poor animal productivity. Research has been carried out to benchmark cow productivity on smallholder dairy herds; however, there is a paucity of information on the current status of breeding practices and the genetic consititution of cattle used in this production system. This information is vital for the development of sound and sustainable breeding programs for SHD production, which can have an enormous positive impact on food security and rural livelihoods. Thus, the aim of this study was to evaluate the levels of genetic diversity and population structure in South African smallholder dairy (SHD) herds using single nucleotide polymorphism (SNP) markers. A total of 192 animals from SHD dairy herds were genotyped using the GeneSeek® Genomic Profiler (GGP) 150K-BeadChip. Four specialized dairy breeds included the Ayrshire(n = 200), Holstein(n = 231), Jersey (n = 224) and Nguni (n = 209) were used as the reference populations. The mean MAF values ranged from 0.30 Ayshire (AYR), Jersey (JER), and Nguni (NGI) to 0.31 Holstein (HOL) and SHD between the populations. There were slight differences in the levels of genetic diversity ranged between 0.39 (JER and NGI) to 0.40 (AYR, HOL, and SHD). A moderate level of inbreeding (0.02) was observed in the SHD population, which results in high genetic diversity among this herds. Principal Component Analysis (PCA) revealed four homogeneous clusters comprising of AYR, HOL, JER, NGI, and a heterogeneous cluster of the SHD. The heterogeneity observed in the SHD population indicates widespread crossbreeding. The model-based cluster analysis corresponded with the PCA and pointed out the predominance of HOL, JER, with marginal gene flow from the AYR and NGI. These results have provided a useful insight into the genetic structure and prevailing breeding practices on South African SHD herds. / National Research Foundation (NRF), Agricultural Research Council (ARC) and University of Limpopo (UL)

Molecular tools, such as sequencing of the mitochondrial DNA Control Region (CR) and genotyping of highly variable nuclear microsatellites were applied to survey the genetic diversity, population structure and phylogeography of three shark species: the whale shark, Rhincodon typus; the bull shark, Carcharhinus leucas; and the nurse shark, Ginglymostoma cirratum. The highly migratory and pelagic whale shark exhibited the largest length variation yet reported for an elasmobranch CR (1143–1332 bp), and high haplotype (h = 0.974 ± 0.008) and nucleotide diversities(π = 0.011 ± 0.006). No geographical clustering of lineages was observed and the most common haplotype was distributed globally. The haplotype frequency, however, differed between the Atlantic and Indo-Pacific populations(AMOVA, ΦST = 0.107, P < 0.001). For the bull shark, both mtDNA CR and five microsatellite loci were surveyed for animals from the Gulf of Mexico, the East coast of Florida and the Brazilian coast. Strong genetic structure was observed between theBrazilian and all northern populations for the CR (ΦST > 0.8, P < 0.001), but not for the nuclear microsatellite. The results here presented are congruent with restricted maternal gene flow between populations as a consequence of female nursery site fidelity. The philopatric tendencies as well as the relatively low levels of genetic diversity raises concerns about the conservation of this species. Finally, for the western Atlantic nurse sharks the genetic diversity estimated in a 1,166 bp fragment of the mtDNA comprising partial cytochrome b, tRNAPro, tRNAThr, and partial CR was the second smallest ever recorded for sharks (h = 0.45 ± 0.04; π = 0.0004 ± 0.0004). The data indicated moderate but significant genetic structure with the mtDNA marker (ΦST = 0.22, P<0.05) and no substantial structure in eight microsatellite loci analyzed. A population bottleneck as recent as the lower Pleistocene might have eroded the nurse shark genetic diversity and also contributed to its relatively lower population structure. The data also indicated that dispersal rather than vicariance better explains the Atlantic distribution of nurse shark, and that the Pacific nurse shark might be a cryptic sister species to Ginglymostoma cirratum.

The world faces severe challenges in providing food security for a growing world population during climate change. This puts pressure on modern agriculture, including adapting crops to new environments and cultivation on less acreage. The tools for adapting crops exist within a species' genetic diversity.  Crop wild relatives (CWR) are wild taxa with a close genetic relationship to our crops. CWRs contain a breadth of genetic adaption for various habitats due to their wide geographical distribution. This invaluable diversity of genes is essential for improving breeding of crops and therefore needs to be sustainably conserved in situ to prevent the loss of the future crop adaptation.  Fragaria vesca appears on the list of priority CWRs for conservation within the Nordic region.  To create an efficient conservation strategy for F. vesca, knowledge about the genetic differences between populations within the Nordic region must be obtained. Therefore, this study aimed to optimise PCR protocol for 10 microsatellite primers in F. vasca. The annealing temperature was successfully optimised for all 10 primer pairs. Two of the primer pairs revealed intra-specific diversity. The study also found support for the earlier discovered genetic divergence between Icelandic and other European populations.

A growing world population means an increase in crop demand. At the same time climate change threatens food security as crops may become maladapted to a new environment. We need to adapt crops to increase crop yield and become resistant to a changed environment to meet this. Crop wild relatives (CWR), which have a genetic relatedness with our crops, will be of importance as a genetic resource for crop adaption and needs to be protected. Vaccinium myrtillus is one of the prioritized species on the Nordic countries CWR priority list. Here I report PCR protocols that can be used for amplifying microsatellites, or SSRs, within V. myrtillus for use in analyses of genetic diversity within and between populations. PCRs with varying annealing temperatures (Ta) were performed. An optimal Ta for the primer pair was found for four SSR loci, and for four SSR loci, an approved Ta was found. These eight primer pairs can be used to amplify SSRs for analyses of the genetic diversity between V. myrtillus populations. This is needed to evaluate which populations to focus on in conservation programmes to ensure a broad genetic diversity usable in crop adaptation to meet increasing crop demand and climate change.

The goal of this study was to examine the taxonomy, ecology, molecular phylogenetics and biosystematics of two apparently closely related species and to elucidate congruent patterns from these often divergent sub-disciplines of systematics. Phylogenetic analysis of DNA sequences showed that Phacelia fimbriata and Phacelia purshii are sister species that demonstrate species-specific differences in germination but little morphologic or genetic differentiation. While a geographic region of sympatry exists, elevation differences preclude co-occurrence. The widespread P. purshii supports higher levels of morphological and genetic diversity compared to the narrow endemic, P. fimbriata. Gene flow between members of this closely related species pair is blocked by hybrid inviability whose expression occurs following normal pollen tube growth and apparent fertilization. Hybrid seeds are inviable, an effect likely caused by an embryo-endosperm incompatibility that, following fertilization, permits development of maternal, but not hybrid fruit and seed tissues. This reproductive barrier adds to our knowledge of an already diverse suite of isolating mechanisms in Phacelia subgenus Cosmanthus.

During the climate oscillations of the Pleistocene an ice sheet formed covering Fennoscandia. Traditionally, Scots pine and many other species were believed to have persisted in glacial refugia in the south, only returning to northern latitudes as the ice retreated. Recent studies have demonstrated the existence of glacial refugia in higher latitudes, in fact, cryptic refugia as far north as Lofoten have been suggested. I investigated the potential existence of such a refugium for Scots pine using mtDNA markers Nad 1-B/C, Nad 7-1 and NODE_new_663. Results show an east/west distribution of the mtDNA polymorphisms at Nad 1 and Nad 7 across Fennoscandia, where multi-locus mitotype bb is more common in the east and ba is more common in the west, in accordance with the hypothesis of a glacial refugium in northeast Europe. Further, the bb mitotype was discovered to be more widespread in Fennoscandia than previously documented. No unique mitotype was discovered in northwestern Fennoscandian populations to support the hypothesis of a glacial refugium along the northwest coast of Norway. Genetic diversity was high and even across Fennoscandia and differences in diversity were not significantly correlated with distance between populations, possibly due to high mutation-rates for the NODE_new_663 minisatellite locus or as a result from admixture between multiple glacial refugia in the area. No conclusive evidence regarding the existence of a glacial refugium in Fennoscandia was discovered and needs to be studied further.

Genotypic diversity within populations can have important evolutionary consequences, but the ecological effects of intraspecific genetic variation on community and ecosystem function have only been studied in a few systems. I present the results of a three-year study designed to address the ecological impacts of genotypic diversity in quaking aspen (Populus tremuloides Michx.), using aspen genotypes planted across genotypic diversity levels (monoculture and mixture) and watering treatment levels (well-watered and water-limited). First, I demonstrated that significant variation exists among genotypes for a wide range of growth, morphological and physiological traits, and quantified high heritability and coefficient of genetic variation values for those traits. This demonstrates that heritable phenotypic variation exists within an aspen population, which could potentially have community and ecosystem implications. Secondly, I collected ground-dwelling arthropods across experimental treatment levels to determine if there are any community-level implications of genotypic diversity and watering treatment. Ground-dwelling arthropods were significantly affected by the genotypic diversity × watering treatment interaction, such that arthropod taxonomic diversity was lowest in water-limited genotypic mixtures. This result runs counter to the bulk of the plant diversity-arthropod diversity literature, which predicts that plant and arthropod diversity should be positively correlated, and highlights the importance of environmental conditions in mediating the plant-arthropod diversity relationship. Lastly, I show that there are no overall effects of genotypic diversity or watering treatment on tree growth patterns. Instead, there are high levels of variation among genotypes in their responses to treatments (significant genotype × diversity × watering treatment interactions), which are often opposing in direction. I also show that there are significant collection site × diversity × watering treatment interactions, demonstrating that genotypes vary in their response to experimental treatments based in part on their original collection site conditions in the field. This study demonstrates that aspen populations contain high levels of genotypic diversity, but that the ecological effects of genotypic diversity are mediated by the environment (in this case, watering treatment) and can be considerably more complicated than found in most previous studies.