Highly sensitive DNA fingerprinting of orchid pollinaria remnants using AFLP
James O. Indsto A C D E , Peter H. Weston A , Mark A. Clements B and Robert J. Whelan CA National Herbarium of NSW, Mrs Macquaries Rd, Sydney, NSW 2000, Australia.
B Centre for Plant Biodiversity Research, National Botanic Gardens, Canberra, ACT 2601, Australia.
C Institute for Conservation Biology, University of Wollongong, Wollongong, NSW 2522, Australia.
D Current address: Westmead Institute for Cancer Research, University of Sydney at Westmead Millennium Institute, Darcy Rd, Westmead, NSW 2145, Australia.
E Corresponding author. Email: james_indsto@wmi.usyd.edu.au
Australian Systematic Botany 18(3) 207-213 https://doi.org/10.1071/SB04009
Submitted: 5 May 2004 Accepted: 21 March 2005 Published: 30 June 2005
Abstract
Numerous Australian terrestrial orchid species in the genus Diuris may be pollinated by food source mimicry. In our field studies, direct observations of orchid–pollinator interactions were rare, but native bees were frequently captured carrying orchid pollinaria, or pollinaria remnants. Sometimes, pollinaria remnants were minimal and included only the viscidium, a sticky pad that was often highly persistent. Confirmation of such tissue as being of orchid source, and attributing them to a particular species can aid pollination studies. DNA-based methods that may identify more or less intact orchid pollinaria are available, but extremely small and degraded samples can pose technical challenges. We have developed an AFLP protocol for such difficult samples that offers some significant advantages over direct PCR-based analysis. We simulated AFLP profiling of very low-DNA samples using DNA template from serial dilutions. A DNA sample range from 6.4 picograms to at least as high as 100 nanograms (15 500-fold range) all yielded AFLP fingerprints. The practical application of this inherent sensitivity of AFLP is demonstrated by the identification of remnants of orchid pollinaria sampled from bees, presented here as a case study. It is expected that this approach will find many applications where sample DNA is limiting, or possibly where pollen of similar appearance may comprise species mixtures.
Acknowledgments
This article is part completion of a Master of Science degree by JI. We acknowledge the financial support of the Joyce Vickery Scientific Research Fund, the Australian Orchid Foundation, and the Herman Slade Orchid Fund. We thank the NSW National Parks and Wildlife Service for permits to complete the study. Jim Mant and Jillian Smith-White from the Royal Botanic Gardens, Sydney provided helpful advice on AFLP. John Riley provided advice on the orchids in the field and Dr Michael Batley provided bee identifications and also supplied the bees containing pollinaria remnants that were used in this study. We also thank Professor David Ayre for critical reading of the manuscript.
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