Orientador: Celso Luis Marino / Resumo: The forest-breeding program in Brazil has the general objective of providing most adapted plants to different environments for various Brazilian regions, for fulfilling timber demands meant for multiple uses in the country. One of the main problems found in different forest breeding programs are the difficulty to identify the different species and hybrids. The use of molecular biology techniques in plant breeding programs is found very effective in the optimization of the time and the direction of these programs, particularly among those plants of the same subgenus. The process of selection and hybrid plants selected for planting in most cases; significantly increase the gain in terms of production and adaptability. The use of molecular markers to characterize the molecular variability of forest species has revolutionized genetic analysis in recent years. The bulk segregant analysis (BSA) is a technique used to identify molecular markers linked to monogenic, dominant or recessive characters. BSA technique in combination with Amplified Fragment Length Polymorphisms (AFLP) technique is an efficient methodology for the detection of polymorphism from genomic restriction fragments through PCR amplification; which helps in analyzing large number of loci for testing without the need for previous information of their sequence in respect to their dominance and reproducibility. The most recent and promising applications of molecular biological methods for the detection of small DNA fra... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

The genetics of colonization is understudied in salamanders but has large conservation implications as new habitats are formed or restored to their previous condition. The 1980 eruption of Mount St. Helens provided a natural experiment to study the genetic effects of a large infrequent environmental disturbance on two species of salamander, Taricha granulosa (Rough-skinned newt) and Ambystoma gracile (Northwestern salamander). Both these species breed in ponds, and are thought to exhibit high breeding site fidelity and low vagility. I designated three treatments based on the effects of the eruption: new ponds (created by the eruption, immigrants only), recovery lakes (in blast zone, survivors plus immigrants), and reference lakes (unaffected by eruption, assumed to represent pre-eruption genetic diversity measures). Salamanders took at least nine years to colonize the new ponds. I studied the population genetics of colonization and recovery using microsatellites and AFLPs (amplified fragment length polymorphisms) to measure genetic diversity, gene flow, and population substructure at Mount St. Helens National Volcanic Monument. Based on population genetics theory and the life history characteristics of these pond-breeding amphibians, I predicted that genetic diversity would be lower in newly colonized ponds compared to recovery or reference sites. I also expected significant levels of population substructuring. Finally, I predicted that because of their lower vagility and large number of neotenes, that A. gracile would have less gene flow and a greater degree of population substructuring than T. granulosa. My predictions were not supported by my data. There was no loss of genetic diversity in new or recovery populations in either species. There was no strong evidence for population substructure by either AMOVA, isolation by distance or principal components analysis. Gene flow (Fst) was high in both species. Taricha granulosa and A. gracile were found to be resistant to a large infrequent environmental disturbance. Loss of genetic variability in new populations cannot automatically be assumed. Predicting dispersal and colonization ability based on the broad category of pond-breeding amphibian is not always reliable.

Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.

Pennisetum Rich, is one of the most important genera in the family Poaceae because it includes forage and crop species such as Pennisetum purpureum Schumach. and Pennisetum glaucum (L.) R. Br. Both P. purpureum and P. glaucum have a number of cultivars and varieties arising due to natural crossing which are very difficult to distinguish morphologically. P. purpureum and P. glaucum also hybridize naturally because they are protogynous and cross pollinated. The resulting hybrids are highly sterile and resemble P. purpureum. Lepidopteran stem borers cause great yield loss in maize produced by resource-poor farmers in Africa and are managed by habitat management or push-pull strategies, in which P. purpureum cultivars and hybrids are used as a trap crop. The aims of this project were to genotype different P. purpureum cultivars and hybrids using Amplified Fragment Length Polymorphism (AFLP) as well as Random Amplified Polymorphic DNA (RAPD) in order to identify cultivars and hybrids and possible misidentifications, assess the congruency of results between AFLPs and RAPDs and to attempt to relate these results to the oviposition preference of Chilo partellus for different P. purpureum cultivars. The individuals to be fingerprinted were collected from several countries in sub-Saharan Africa, a few from the USA and one from China. The AFLP analysis of these individuals were done with primer combinations EcoRI/MseI and Mlul/Msel on polyacrylamide gels and an ABI 3130 xl Genetic Analyzer respectively. The automated sequencer visualized more bands than the polyacrylamide gels. The RAPD technology was not developed any further after 17 primers were tested and no polymorphic bands detected. Overall results indicated that cultivars did not cluster according to geographical origin, and cultivars known by popular names did not always cluster together, indicating diversity within the cultivar or misidentifications. An example of a misidentification is the cultivar Green Gold being no other than cultivar Harare, or cultivar Swaziland 3 being cultivar Sanitas. Proper management by nursery managers cannot be stressed enough, as this will prevent plants getting mixed up, causing confusion. There was no relationship between the relatedness of cultivars and moth oviposition preference. The AFLP technology could be a powerful tool for the DNA fingerprinting and molecular characterization of this grass species, but poor germ plasm management negates its application. / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2008.

Total antioxidant activity and total carotenoid levels were evaluated for more than 100 common potato (Solanum tuberosum, L.) cultivars grown in the United States, advanced breeding lines from several Western U.S. breeding programs, and 47 related, tuber-bearing species. An initial assessment of variability for antioxidant activity provided baseline information to be used for potential potato promotion and for the development of new varieties with greater human health benefits. Wide variability in antioxidant levels provided evidence of genetic control of this trait, indicating that it could be possible to breed for enhanced levels of antioxidant compounds in potato. Accessions, varieties, and advanced breeding lines identified in the broad screen as having high antioxidant activity and high total carotenoid levels, were fine screened via HPLC to determine specific phenolic and carotenoid compounds present in potato. The objective of the study was to identify parents for use in the Texas breeding program to develop potato varieties containing increased levels antioxidant compounds. In the broad screen for total antioxidant activity, the 47 related, tuber-bearing species showed a wider range of variability than the cultivated varieties and breeding lines. Based on the DPPH assay, antioxidant activity ranged from 103-648 uM trolox equivalents in the cultivated varieties and advanced breeding lines, while that of the wild species was 42-892. HPLC analysis revealed that the phenolic content of the species, and their cultivated counterparts, was primarily composed of caffeic and chlorogenic acids. Other phenolics identified were p-coumaric acid, rutin hydrate, vanillic acid, epicatechin, t-cinnamic acid, gallic acid, and salicylic acid. The highest phenolic content discovered in the accessions was five-fold higher than the highest of the cultivated genotypes. Carotenoid analysis revealed lutein in the accessions, but the yellow-flesh breeding lines were much higher in carotenoids. In addition to the work conducted on antioxidants, an attempt was made to separate intraclonal variants of the potato cultivar Russet Norkotah. Eleven microsatellite primers and 112 AFLP primer combinations failed to produce any reproducible polymorphisms. The inability to detect differences between the clones could be due to the tetraploid nature of the clones or epigenetic differences not detected by the procedures utilized in this study.

Lately the rate of genetic gain in most agronomic crop species has been reduced due to several factors that limit breeding efficiency and genetic gain. New genetic tools and more powerful statistical analyses provide an alternative approach to enhance genetic improvements through the identification of molecular markers linked to genomic regions or QTLs controlling quantitative traits. The main objective of this research was to identify genomic regions associated with enhanced agronomic performance in lines per se and hybrid combination in Sorghum bicolor (L.) Moench. A population composed of 187 F5:6 recombinant inbred lines (RIL) was derived from the cross of restorer lines RTx430 and RTx7000. Also, a testcross hybrid population (TCH) was developed by using each RIL as a pollinator onto ATx2752. A linkage map was constructed using 174 marker loci generated from AFLP and SSR primer combinations. These markers were assigned to 12 different linkage groups. The linkage map covers 1573 cM with marker loci spaced at an averaged 9.04 cM. In this study, 89 QTL that control variation in seven different morphological traits were identified in the recombinant inbred line population, while in the testcross hybrid population, 79 QTL were identified. These traits included grain yield, plant height, days to mid-anthesis, panicle number, panicle length, panicle exsertion and panicle weight. These putative QTL explained from 4 to 42% of the phenotypic variation observed for each trait. Many of the QTL were not consistent across populations and across environments. Nevertheless, a few key QTL were identified and the source of the positive additive genetics isolated. RTx7000 was consistently associated with better agronomic performance in RIL, while in testcrosses, RTx430 was. Some genomic regions from RTx7000 may be utilized to improve RTx430 as a line per se. However, it is very unlikely that such regions will have a positive effect on the combining ability of RTx430 since testcross results did not reveal any transgressive segregants from the RIL population.

<i>Mycoplasma bovis</i> is generally considered the causative pathogen associated with Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in feedlot cattle. However, <i>M. bovis</i> virulence may vary between strains as it is also isolated from asympytomatic cattle. The following study aims to determine the prevalence of <i>M. bovis</i> in the respiratory tract of western Canadian cattle using two sampling methods and at two time points following feedlot entry. Three study groups were sampled. In the first group nasal swabs (NS) and bronchoalveolar lavages (BAL) were taken from 36 clincally healthy cattle at the University of Saskatchewan feedlot at both 14 and 90 days on feed (DOF). In a second experiment, NS were taken from 56 animals upon arrival at a commercial feedlot and one week to three months later upon treatment for respiratory disease. Lung and joint tissue swabs were collected at necropsy from a third group of 19 animals with CPPS clinical pathology originating in 10 different western Canadian feedlots. All samples were selectively cultured for <i>Mycoplasma</i> spp. DNA was extracted from isolated putative <i>Mycoplasma</i> colonies and amplified with universal 16S rRNA gene primers for identification. Amplified Fragment Length Polymorphism (AFLP) was used to genetically differentiate <i>M. bovis</i> positive isolates. More <i>M. bovis</i> was isolated from NS than BAL and <i>M. bovis</i> prevalence increased with DOF in the feedlot in both the University of Saskatchewan and commercial feedlot trials. Three genetically distinct clusters (A, B, and C) were isolated from the necropsy group. Two of these clusters were primarily associated with isolates collected from feedlot cattle and one strain was exclusively found in CPPS-associated mortalities. No significance difference in the prevalence of <i>M. bovis</i> strains was observed between different days on feed or sampling methods. It was concluded that either the difference in disease state is a host dependent outcome, due to a multi-factorial disease complex, or the AFLP assay was not sensitive enough to differentiate strains based on virulence.

<i>Mycoplasma bovis</i> is generally considered the causative pathogen associated with Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in feedlot cattle. However, <i>M. bovis</i> virulence may vary between strains as it is also isolated from asympytomatic cattle. The following study aims to determine the prevalence of <i>M. bovis</i> in the respiratory tract of western Canadian cattle using two sampling methods and at two time points following feedlot entry. Three study groups were sampled. In the first group nasal swabs (NS) and bronchoalveolar lavages (BAL) were taken from 36 clincally healthy cattle at the University of Saskatchewan feedlot at both 14 and 90 days on feed (DOF). In a second experiment, NS were taken from 56 animals upon arrival at a commercial feedlot and one week to three months later upon treatment for respiratory disease. Lung and joint tissue swabs were collected at necropsy from a third group of 19 animals with CPPS clinical pathology originating in 10 different western Canadian feedlots. All samples were selectively cultured for <i>Mycoplasma</i> spp. DNA was extracted from isolated putative <i>Mycoplasma</i> colonies and amplified with universal 16S rRNA gene primers for identification. Amplified Fragment Length Polymorphism (AFLP) was used to genetically differentiate <i>M. bovis</i> positive isolates. More <i>M. bovis</i> was isolated from NS than BAL and <i>M. bovis</i> prevalence increased with DOF in the feedlot in both the University of Saskatchewan and commercial feedlot trials. Three genetically distinct clusters (A, B, and C) were isolated from the necropsy group. Two of these clusters were primarily associated with isolates collected from feedlot cattle and one strain was exclusively found in CPPS-associated mortalities. No significance difference in the prevalence of <i>M. bovis</i> strains was observed between different days on feed or sampling methods. It was concluded that either the difference in disease state is a host dependent outcome, due to a multi-factorial disease complex, or the AFLP assay was not sensitive enough to differentiate strains based on virulence.

Human-induced eutrophication is indirectly affecting aquatic organisms by altering their environment. This brings on altered selective pressures and could thereby cause changes in the genetic composition of exposed populations. Since anthropogenic environmental changes are usually occurring at a much higher rate than naturally occurring changes, they force populations to adapt to the new conditions faster than normal. Here, I have studied populations of three-spined sticklebacks (Gasterosteus aculeatus) from four eutrophicated and four adjacent reference sites, along the coast of Finland, to investigate if this species has responded genetically to the human-induced eutrophication of the Baltic Sea. For this purpose I used amplified fragment length polymorphism (AFLP) and found distinctions in genetic composition between the two habitats, as well as similarities between populations from eutrophicated sites. This suggests a similar genetic response to eutrophicated conditions by stickleback populations from different geographical areas. Moreover I found a distinct geographic structure among three-spined sticklebacks in the Baltic Sea.

Human-induced eutrophication is indirectly affecting aquatic organisms by altering their environment. This brings on altered selective pressures and could thereby cause changes in the genetic composition of exposed populations. Since anthropogenic environmental changes are usually occurring at a much higher rate than naturally occurring changes, they force populations to adapt to the new conditions faster than normal. Here, I have studied populations of three-spined sticklebacks (Gasterosteus aculeatus) from four eutrophicated and four adjacent reference sites, along the coast of Finland, to investigate if this species has responded genetically to the human-induced eutrophication of the Baltic Sea. For this purpose I used amplified fragment length polymorphism (AFLP) and found distinctions in genetic composition between the two habitats, as well as similarities between populations from eutrophicated sites. This suggests a similar genetic response to eutrophicated conditions by stickleback populations from different geographical areas. Moreover I found a distinct geographic structure among three-spined sticklebacks in the Baltic Sea.