Saving An Endangered Species of the Lady's Slipper Orchid!

AJAS · 2022 Plant Science

Kelly Salmon, Laura Schaner

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Overview

The Cypripedium (Cyp.) genus of terrestrial orchids is limited to a narrow range of climates and habitats throughout the world. Climate change related habitat loss has profound implications on population survival of Cypripedium species. Robust genetic diversity within a Cypripedium species may allow for adaptive responses to environmental stresses such as climate change. Understanding Cypripedium genetic diversity may facilitate strategies to improve population survival. Cypripedium reginae (showy lady’s slipper), which is the focus species of this project, is a threatened species local to New Hampshire and Vermont. Microsatellite primers were found by others to amplify regions of the Cyp. tibeticum genome in order to assess genetic diversity. The primary goal of this project is to examine whether these primers could effectively bind to DNA sequences from Cyp. reginae samples and lead to amplification of microsatellite regions. Primers that successfully amplified microsatellite regions could subsequently be used for an analysis of genetic diversity among individual plants within fens as well as among fens. DNA samples were first extracted from Cyp. reginae leaves from an artificial fen in Lyme, New Hampshire. PCR and gel electrophoresis were then used to determine whether each primer set amplified DNA from Cyp. reginae. Successful reactions were purified and sent for DNA sequencing. Of the 10 microsatellite primer sets tested, four produced clear bands in the gel electrophoresis stage. After sequencing, only two of the isolated DNA samples were homologous to the original Cyp. tibeticum microsatellite sequences. These findings demonstrate that at least two of the ten analyzed primers can effectively assess genetic variation in Cyp. reginae. The future of this study will involve an evaluation after all primers have been re-tested, the remaining Cyp. tibeticum primers have been tested, and more sequences have been acquired. Further analysis will include an assessment of genetic diversity within and among fens using the successful primers.

Video

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From the student

My story begins as a seventh grader when I first decided to participate in an afterschool program at the New Hampshire Academy of Science. I saw it as an opportunity to spend my afternoons learning basic laboratory techniques and be part of a project I found interesting. After two years of this program, I attended the summer program at the NHAS in 2020, where I developed my first individual project, testing the water quality at a nearby lake. Although this session was mainly over zoom due to the coronavirus pandemic, I was still able to have a wonderful experience! Throughout the following school year, I worked with the bacteria I collected from the water, giving me more experience with research and developing projects. My current project is from the 2021 summer program, where I decided to diverge from my water quality path and focus on something different. I found the Lady's Slipper project options to be extremely interesting, and over the course of three weeks, I tested microsatellite primers on samples of the Showy Lady's Slipper in order to assess whether these primers could be used in an analysis of genetic diversity within fens in New Hampshire and Vermont. Using my findings, I wrote a summary paper and the subsequent events landed me here! I'm really looking forward to 2022's AJAS conference!

References

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Bebeau, G. “Showy Lady’s-Slipper.” The Friends of the Wild Flower Garden, Inc. Plants of the Eloise Butler Wildflower Garden The Oldest Public Wildflower Garden in the United States, 2015, www.friendsofthewildflowergarden.org/pages/plants/showyladysslipper.html.

Grant, Bonnie. “Harvesting Lady Slipper Seed Pods – How To Collect Lady Slipper Seeds.” Gardening Know How, www.gardeningknowhow.com/ornamental/flowers/orchids/lady-slipper-seed propagation.htm#:~:text=Lady%20Slipper%20Seed%20Germination, Lady%20Slipper%20orchids&text=The%20orchid%20blooms%20April%20through,a 20natural%20soil%2Dborne%20fungus.

“FinchTV.” Digital World Biology, digitalworldbiology.com/FinchTV.

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Biolabs, New England. “Protocol for Extraction and Purification of Genomic DNA from Tissues (NEB #T3010).” NEB, www.neb.com/protocols/2018/10/24/protocol-for extraction- and-purification-of-genomic-dna-from-tissues-t3010.

NHGRI. “Polymerase Chain Reaction (PCR) Fact Sheet.” Genome.Gov, 9 Mar. 2019, www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet.

“Lect. 8. Intro. to Microsatellites.” Genetic Markers, ZOO 4425/5425, Fall 2008, www.uwyo.edu/dbmcd/molmark/lect08/lect8.html. Accessed 29 June 2021.