Thesis (Ph.D.)--Victoria University (Melbourne, Vic.), 2009.

published_or_final_version / Statistics and Actuarial Science / Master / Master of Philosophy

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DNA fingerprinting was applied for the molecular elucidation of taxonomic relationships within a genus of orchids which have previously been based on morphological characteristics. Phragmipediwn consists of 15-20 species native to Central and South America. This research project included two studies. In the first study DNA was isolated from 11 samples (including two unidentified ones). These individuals, which were mostly hybrids, were found in the Wheeler Orchid Collection and Species Bank at Ball State University. In order to position Phragmipediwn within the orchid family fingerprinting was also performed on individuals in the sister taxa, Cypripedium and Paphiopedium, which are members of the same subfamily, and on a member of the outgroup taxon Vanda. The polymerase chain reaction (PCR) was employed to yield fingerprints resulting from the use of random primers. Fifty nine random amplified polymorphic DNA (RAPD) bands were obtained using 5 different primers to yield 107 polymorphic bands. As many as 75% of genetic loci were found to be shared between hybrids that resulted from a cross of more than one individual in the same section. However the percentage dropped to 35-65 % when only one parent was shared in the cross. Furthermore, the sister group taxa Cypripedium and Paphilopedium shared from 12 % -35 % of their polymorphic loci with the members of the genus Phragmipedium. The outgroup taxon Vanda shared 17% of its polymorphic loci with the rest of the samples.In a second study DNA was isolated from one member of each of the five sections of the genus Phragmipedium, and RAPD-PCR fingerprinting was used to compare their genetic similarities to that of the two sister taxa and the outgroup taxon. It was found that individuals in different genera shared 25% or less of their polymorphic bands. Between sections of the same genus 20-50% of genetic loci were shared. Two sections, Platypetalwn and Phragmipedium showed the highest degree of genetic relatedness (41-53%). Again the outgoup taxon shared less than 20% Phragmipediwn samples on the phenograms produced but the percentage was again insignificant. However, genetic analyses of the members of the section Lorifolia gave conflicting results: 46% genetic identity was observed in the first trial and 20% in the second.In conclusion, RAPD-PCR fingerprinting results appeared to be effective in the positioning of sections within a genus indicating the degree of similarity of closely related taxa. Also RAPD-PCR was able to place an unknown individual within a specific section of the genus. However, it could not be employed to determine the identity of unknown species due to the high degree of genetic diversity observed between even closely related individuals. / Department of Biology

Degree awarded with distinction on 6 December l995. A dissertation submitted to the Faculty of Science, University the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host liver cell DNA is associated with the development of hepatocellular carcinoma (HCC). This is indicated by epidemiological trends, molecular studies and studies of animal models infected with viruses closely related to HBv. However, little is known about the mechanism by which the integrated HBV DNA includes HCC despite continuing analysis of the integrated HBV DNA and its surrounding cellular sequences. [Abbreviated Abstract. Open document to view full version]

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Given that morphological identification of willow is difficult, willow lines being investigated for their suitability for use as short rotation crops for biomass production in Saskatchewan were investigated with various molecular techniques as possible tools for DNA fingerprinting. Flow cytometry was used to assess variation in nuclear DNA content and thus ploidy level of the lines of the five species (<i>Salix purpurea, Salix eriocephala, Salix sachalinensis, and Salix dasyclados</i>) and three hybrids (<i>S. purpurea x S. miyabeana, S. sachalinensis x S. miyabeana, S. viminalis x S. miyabeana</i>). The DNA content varied between 1.14 and 3.00pg. Ploidy levels of the species varied from triploid to hexaploid while all hybrids were tetraploid. RAPD and ISSR marker systems were used to assess genetic and taxonomic relationships among all willow lines. Of 90 RAPD primers tested, 60 were selected and 99 polymorphic bands scored. Of 35 ISSR primers tested, 19 were selected and 35 polymorphic bands scored. Both RAPD and ISSR dendrograms clustered together lines belonging to the same species and same hybrid combination. A combination of strong and reproducible RAPD and ISSR bands was used to develop identification keys for lines belonging to the same species.<p> The ribosomal RNA gene region, including the entire 5.8S RNA gene and the internal transcribed spacers (ITS1 and ITS2) was amplified and sequenced to assess sequence homology between the five species. The total length of the amplified region was 601bp, with the ITS1, 5.8 S and ITS2 being 223, 163, and 215bp respectively. Intra- and inter-species SNPs were observed, 6 within ITS1, and 3 within ITS2. No polymorphisms were found in the 5.8S gene. The low rate of variation within the sequenced ITS fragment between species supports the monophyly of the five species involved in this study, and confirms their belonging to the subgenus Caprisalix. SCAR primers were designed from species-specific polymorphic nucleotides and applied to the willow collection to test their use for species identification. A species identification key based on SNPs is proposed.