Establishment of phylogenetic relationships within the genus Phragmipedium using RAPD-PCR fingerprinting

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Micha, Caterina
Vann, Carolyn N.
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Thesis (M.S.)
Department of Biology
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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.