This research was an attempt to identify and provide a phylogeny of Vanilla orchids using PCR sequencing/ fingerprinting.Traditional classification methods haved relied heavily on morphological traits and are often contradictory, subjective and incomplete. This research attempted to discover an objective and reliable method for identification and classification of selected tropical orchids species using molecular techniques. Vanilla orchids were chosen for their commercial importance in the scent and flavoring industry.Reconstruction of phylogenetic data is possible using relatively new molecular techniques: Polymerase Chain Reaction (PCR) fingerprinting and sequencing. PCR fingerprinting reveals restriction fragment length polymorphisms (RFLPs) within an organism's genome. Thesevariations can be used to construct genetic maps in a variety of species. PCR sequencing reveals genetic variation at an even greater level: nucleotide sequence.The literature suggests that these methods are fairly quick and simple; however, the crucial step is obtaining high molecular weight DNA digestable with restriction enzymes or amplifiable by the PCR. DNA isolation has proven to be difficult with Vanilla orchids due to their numerous phenolic compounds, tough fibrous tissue and high number of polysaccharides. This research developed a method of isolating high molecular weight DNA from orchids. This DNA was digestible with restriction enzymes. The DNA was subjected to DNA fingerprinting with primers specific to the 18S-26S ribosomal RNA gene and with RAPD primers. The DNA was also sequenced using the PCR technique.Variation between Vanilla species genomes was discovered and compared to traditional phylogenetic information. It was found that banding patterns and nucleotide sequences were almost identical for Vanilla planifolia and planifolia varegata. The banding patterns for Vanilla barbellata were similar to those of the two unknown Vanilla orchids. Vanilla humboltii exhibited a banding pattern far different from either barbellata or planifolia. These results confirm the hypothesis that PCR fingerprinting/ sequencing is a useful technique for the identification and phylogenetic analyses of Vanilla orchids. / Department of Biology
| Identifer | oai:union.ndltd.org:BSU/oai:cardinalscholar.bsu.edu:handle/184963 |
| Date | January 1994 |
| Creators | Sanborn, Kristi L. |
| Contributors | Ball State University. Dept. of Biology., Vann, Carolyn N. |
| Source Sets | Ball State University |
| Detected Language | English |
| Format | ix, 83 leaves : ill. ; 28 cm. |
| Source | Virtual Press |
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