Testing Microsatellite Primer Sets On Cypripedium Arietinum

Kelly Salmon, Advika Govindaraju

Overview

Cypripedium arietinum, or ram’s head lady’s slipper orchids, are endangered in New Hampshire and threatened in Vermont. A genetically diverse population will be more able to adapt to a changing environment than a less genetically diverse population. It is unknown how genetically diverse Cyp. arietinum populations are in NH and VT. This investigation tested the efficacy of microsatellite primers developed for Cyp. tibeticum on Cyp. arietinum and utilized the successful primers to analyze the genetic diversity in a single Cyp. arietinum population in Vermont. Microsatellites are short regions of DNA repeats which accumulate mutations at a higher rate than other regions of a given genome. Cyp. arietinum leaf samples were collected from a small population in a forest in Strafford, VT. DNA was extracted from leaves taken from three different plants. DNA samples were amplified with 10 Cyp. tibeticum microsatellite primer sets with polymerase chain reaction. Gel electrophoresis was conducted to determine if the primers amplified Cyp. arietinum DNA. Five of the 10 primers were shown to effectively amplify DNA. The amplified DNA samples were purified and sent to an external lab for DNA sequencing, yielding chromatograms for analysis. The chromatograms were analyzed for double peaks that represent heterozygous positions. The DNA sequences of all three samples were shown to be similar. Since only three plants were tested, there is not enough data to confirm the diversity among these plants in the local population. Future research will involve using additional primers for genetic analysis with more samples from different plants in order to draw accurate conclusions about the genetic diversity of this species in Vermont and New Hampshire.

From the student

I applied for the New Hampshire Academy of Science research program during the summer of 2021. Out of all the project options, I was interested in studying Lady's Slipper Orchids. It was my first time doing something like this and I was unsure about how it would go but I decided to try it. Once I began my project I learned how to do PCR, gel electrophoresis, and so much more during the 3 week program. I was part of a hybrid group and worked from home a few days every week. Although I had to remember to keep on bringing supplies to and from the lab, I was able to conduct just as much research from home as I was at the lab. My wonderful mentor, Dr. Kelly Salmon, assisted me with my research and was always there to answer my never ending questions throughout the entire process. Dr. Peter Faletra and Dr. Alyson Michael were also very supportive during my time there.

I really enjoyed learning so many new things and getting hands on experience. I'm excited to have the opportunity to present my work at AJAS and am looking forward to learning about other projects and exploring other events at the conference.