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Abstract Detail


Systematics Section / ASPT

Comer, Jason [1], Rettig, Jeff H. [2].

Evaluation of eight protocols for PCR thermocycling of the internal transcribed spacer region in Aeranthes Lindley (Orchidaceae).

Orchidaceae is one of the largest families with approximately 24 000 species; morphological diversity makes taxonomic studies difficult. The internal transcribed spacer (ITS) region of nrDNA is used extensively because for phylogenetic studies it is easily amplified, small in size, and is variable enough. Most protocols for PCR amplification of the ITS region follow those described for Sorghum or the Asteraceae. For this study eight different protocols from seven studies using the ITS region to evaluate orchid phylogenetics were selected to evaluate the effectiveness of ITS amplification in the genus Aeranthes. The PCR reaction mixtures used were based on the mixtures from the evaluated papers. Two external forward primers, one external reverse primer and two (forward and reverse) internal primers were included. Amplified PCR products were visualized on a gel, purified and cycle sequenced. Sequencing results were compared to published sequences to confirm the desired region was amplified. The efficiency of each protocol was determined by band intensity, minimal additional (multiple) banding in the gel and conformation that the correct segment was amplified. Pridgeon et al.s (subtribe Pleurothallidinae) protocol was the most effective in this study. The next effective was Carlsward et al.s (tribe Vandeae including Aeranthes) protocol for the full ITS amplification followed by Carlsward et al.s (tribe Vandeae) protocol for the amplification of part of the ITS region. Van den Berg et al.s (subfamily Epidendroideae), Gravendeel et al.s (Colelogyne), and Koehler et al.s (Bifrenaria) protocols were not as effective. Douzery et al.s (tribe Diseae) protocol was minimally effective and Whitten et al.s (tribe Maxillarieae) protocol was not effective in amplifying the Aeranthes ITS region.


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1 - College of the Ozarks, Biology, P.O. Box 17, Point Lookout, Missouri, 65726, USA
2 - College of The Ozarks, Department of Biology, Po Box 0017, Point Lookout, Missouri, 65726, USA

Keywords:
Orchidaceae
Aeranthes
ITS
PCR
Thermocycling
Protocol Evaluation.

Presentation Type: Poster:Posters for Sections
Session: P
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM
Number: P59070
Abstract ID:1877


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