This diploma thesis is focused on optimalization and employing the PCR-RFLP method, based on the molecular biology principles, for an identification and taxonomy of the yeasts which colonize the leaf surfaces. Simultaneously the yeasts identification techniques based on physiological and morfphological attributes are compared and replaced. PCR-RFLP takes advantage of thermostable polymerases´ ability to amplify the specific segment in the rDNA, which can be split by restriction endonucleases to characteristical polymorphical fragments. Comparing these fragments and restriction´s positions which are for each species unique, demanded results were obtained. They´re summarized in the conclusion part. The theoretical part describes the morphology and cytology of the yeasts, taxonomy as a science, genuses of examined yeasts Cryptococcus, Rhodotorula a Saccharomyces are covered more thoroughly and the method of PCR-RFLP is described in detail.

This diploma thesis has analyzed the effect of organic wine-growing on the wine yeasts population. The wine yeasts were isolated from the Pinot Noir variety. They were identified by the molecular biological method PCR-RFLP. The theoretical research compiles basic information on yeasts, knowledge about the red wine production as well as information on molecular biological methods. The experimental part utilizes the 5,8S-ITS rDNA specific segment for analysis. The segment was amplified using the ITS1 and ITS4 primers and subjected to restriction analysis. The restriction analysis has used these restriction endonucleases - HaeIII, HinfI, Taq?I, AluI and MseI. The BioNumerics software was then used to compare genetic similarity between the isolated yeasts and these were taxonomically classified.

The aim of this diploma thesis is the identification of yeasts isolated during grape must fermentation. The must was obtained from Pinot Noir varieties grown in an integrated and organic production. The partner of this thesis was a winery Holánek. In the theoretical part of the work was the emphasis on information about the determinants quality of wine, yeasts and PCR-RFLP method. Physiological properties of yeasts were described and also the principles of the polymerase chain reaction (PCR) were explained. In the experimental part of the thesis was applied molecular biology method PCR-RFLP for identification of yeasts. The specific segment of DNA was amplified (5, 8S-ITS rDNA sequencing) with the help of ITS1 and ITS4 primers. The incurred amplicons were digested by applying restriction endonucleases: HaeIII, HinfI and TaqI. Subsequently the restriction fragments were analysed by using of electrophoresis. The yeasts were identified and classified by taxonomy on the level of genera and species.

This diploma thesis focuses on isolation and taxonomic classification of yeast species isolated during the red wine (Pinot noir) fermentation. Grapes were grown under organic and integrated farming in South Moravia wine region, Czech Republic. Processing was controlled – for inoculation was used strain Saccharomyces cerevisiae BS6. Polymerase chain reaction followed by restriction fragment lenght polymorphism of PCR-amplified fragments (PCR-RFLP) was used for yeast species identification. For DNA analysis we used coding region of 5.8S ITS rDNA which was amplified using ITS1-ITS4 primers. Amplicon was digested by three restriction endonucleases - HaeIII, HinfI and HhaI. Isolates were divided into eleven groups using UPGMA cluster analysis (software BioNumerics). We identified following yeast species: Candida valida, Candida vini, Issatchenkia occidentalis, Pichia fermentans, Saccharomyces cerevisiae and Zygosaccharomyces bailii. We were not able to identify some yeast species. Differences between organic and integrated farming were demonstrated with varying composition of yeast species.

This thesis deals with identification of yeasts by applying the RFLP-PCR method. Objective of the thesis was to identify the yeasts present in wine from Grüner Veltliner during fermentation. Identification was made by amplification of 5,8S-ITS sequences of DNA by the polymerase chain reaction with primers ITS1 and ITS4. Amplified DNA was submitted to the restriction analysis by restriction endonuclease HaeIII, HinfI and HhaI. By restriction analysis with a specific enzyme, the amplified DNA is chopped into the specific fragments which are characteristic for given kind of yeasts. In the analysed wine, the dominance of autochthonal yeast Saccharomyces cerevisiae was confirmed throughout fermentation. The other identified yeasts in the wine were of kind Pichia. The second part of the thesis was to expand the database by characterization of 28 type-yeasts, using RFLP-PCR analysis. To compare the genetic similarity, program BioNumerics was used, which processed the results of UPGMA cluster analysis using Jaccard´s coefficients.

The aim of this thesis is the isolation and identification of yeasts obtained from the wine berries and the characterization of the collection yeast by using processes of PCR - RFLP. The type yeasts were obtained from the collection of yeasts of CCY in Bratislava, yeasts from the wine berries were collected from the species of Hibernal wine from the wineries of Štěpán Maňák. Identification of individual yeast is then based on analysis of the DNA segment in the area of 5,8S - ITS using primers ITS1 and ITS4. The restriction analysis was performed using restriction endonucleases HaeIII, HinfI, HhaI a TaqI(a). Restriction analysis is used to chopp the DNA to specific sections that are characteristic for each microorganism. For the assesment of the genetic similarity analyzed yeasts the BioNumerics software has been used. BioNumerics processes the results using cluster analysis using Jaccard´s coefficients.

Lichens are defined as symbiotic associations composed of a fungal partner, the mycobiont, and one or more photosynthetic partners, the photobiont (1). A currently employed method for the identification of photobionts is the culture of photobiont from the lichen, but this method employs a labor intensive and long cultivation period, thus identification has been neglected. Out of the approximatelyl4,000 lichen described, only about 4% oflichen photobionts have been identified to species (1). In this study we investigated the feasibility of developing a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) identification system for rapidly identifying lichen algae of the genus Trebouxia (In this study we consider Pseudo-Trebouxia part of the genus Trebouxia). DNA was isolated and purified from cultures of each Trebouxia species. A 1300 hp fragment of the 5' region of the nuclear-encoded large subunit (26S) ribosomal RNA genes was amplified by PCR (2). This 5'region of the 26S region is considered to be a byper-variable region because it differs amongst Trebouxia (2) making it a good candidate for RFLP. The sequences were then analyzed with restriction analysis software to determine restriction maps and individual virtual RFLP patterns. Patterns were constructed using the program SPR Opt (SNP and PCR-RFLP Optimization) allowing each Trebouxia species to be identified by a distinctive restriction pattern. We were unable to validate the key due to contamination of materials, which lead to inconclusive data. Future experiments aim to validate the key by comparing the virtual RFLP patterns to the actual patterns obtained for each type culture of each species.

No description available.

Soybean mosaic virus (SMV) is the major virus disease reported all over the world in soybean crop. This disease causes reduction in the yield and quality of soybean crop. Three independent genes Rsv1, Rsv3, and Rsv4, were found to provide host resistance in soybean. Rsv1 confers resistance to all but most virulant strains of SMV. Rsv1 has been mapped to soybean molecular linkage group (MLG) F by using molecular markers. The purpose of this study is to investigate the location of Rsv3 gene on soybean map using molecular markers. The Rsv3 gene of soybean confers resistance to the most vurulent strains (G5-G7) of SMV. In order to map the gene, an F2 population was constructed from a cross between L29, an Rsv3 isoline of 'Williams', and 'Lee 68', a susceptible cultivar. Rsv3 genotypes of 183 F2 plants were determined by inoculating F2:3 progeny with the G7 strain of SMV. A preliminary survey of two parental lines, near isogenic lines (NILs), and bulk segregants with 136 restriction fragment length polymorphism (RFLP) markers yielded 36 markers showing variation between the two parents. These polymorphic RFLP markers unable to provided any indication of linkage to Rsv3. As an alternative strategy, amplified fragment length polymorphic (AFLP) marker analysis of the two parental lines, NILs and bulk segregants was performed using 64 primer combinations. Initial breakthrough came in the form of AFLP primer combination of Eco+AAC/Mse+CTG exhibited polymorphism between NILs, bulk segregants, and two parental lines. This AFLP marker was isolated and cloned to convert it into a RFLP clone to further investigate the linkage to Rsv3 by F2 segregation analysis. A mapping population constructed by crossing Glycine max x Glycine soja employed in determining the location AFLP-derived RFLP clone on soybean linkage map. This population has densely mapped molecular marker data that enabled determining the location of AFLP-derived RFLP clone ACR1 on soybean molecular linkage group (MLG) B2 between the markers pA516 and pA519. This finding, made it easy to establish the linkage of markers pA519, pA516, and pA593 in L29 x Lee 68 population by F2 segregation analysis. The closest marker linked pA519, was 0.9 cM away from Rsv3. In another study Rsv4 is reported to be mapped to MLG D1b of soybean. Results of this study are useful in marker-based selection (MAS), pyramiding viral resistance genes and in cloning the Rsv3 gene. / Master of Science

Intestinal flora of wild chimpanzee has not been studied. Fecal flora analyses currently give insight to this environment. We collected feces from twelve human-habituated wild chimpanzees in each of three age groups: four juveniles, four sub-adults, and four adults. We analyzed fecal samples using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) of amplified 16S rRNA genes to determine bacterial diversity present. Between 1 and 14 terminal-restriction fragments (T-RFs) were observed in each sample. A total of 26 unique T-RFs were produced from the samples and ranged in size from 92 to 837 base pairs (bps). Twenty-four of these T-RFs corresponded to five bacterial phyla: Actinobacteria, Bacteroidetes, Firmicutes, Mollicutes, and Proteobacteria, as well as uncultured and unidentified bacterial species. The remaining T-RFs corresponded solely to uncultured or unidentified bacteria. Firmicutes was the most common phylum, observed in 11 of the samples. Bacteroidetes was the second-most common phylum, detected in 8 of the samples. Principal Components Analysis (PCA) revealed a discrete clustering of 10 samples when looking at components one and two, and a clustering of 11 samples when looking at component three. These three components accounted for 72.5% of the variation within the data. Morisita indices were computed to compare T-RF profiles of two samples at a time, and were between 0 and 0.886. Results indicated that some fecal bacterial profiles were similar in the study group, but ultimately varied between samples when compared two at a time. Specific diet, physiology, and environmental reservoir exposure may play large roles in shaping such profiles. / Master of Science