Genetic Diversity in Microsatellite Regions of Cypripedium parviflorum

Tiffany Sun

Overview

Many of the 58 species of the Cypripedium orchids located around the world are being increasingly threatened with extinction, largely because of habitat degradation or habitat loss. An initial step to inform plant species conservation is to assess the genetic diversity of a population to gauge the population’s likelihood for survival under environmental stresses. In this project, genetic diversity was assessed by examining microsatellite regions, which are short repetitive sections of the DNA that mutate more frequently than many other genomic regions. A goal of this research is to test the effectiveness of primers developed by another group for the Cyp. tibeticum lady’s slipper orchid on Cyp. parviflorum (yellow lady’s slipper) to determine which primers bind to the DNA successfully. The primers that produce positive results yield amplified DNA that can be sequenced to assess genetic diversity among Cyp. parviflorum samples. Five leaf samples were collected from Cyp. parviflorum plants in Strafford, VT. All 5 samples underwent the processes of DNA extraction, PCR, gel electrophoresis, and DNA cleanup in preparation for sequencing. Our current hypothesis is that plants that are nearby to each other will show less genetic diversity than plants that are a farther distance apart (in different clumps). This conjecture was based on the understanding that plants that are nearby to each other (1/4 meter) may have grown from vegetative (asexual) reproduction and be genetically identical to the parent plant. Plants that are farther apart (more than 5 meters) likely have grown from seeds produced through sexual reproduction and will possess greater genetic diversity. To assess genetic diversity, primer sets that reliably amplify microsatellite regions need to be identified to sequence these variable regions. Ten primer sets were tested and, among them, six produced consistent clear positive results in successfully copying the DNA of interest. This did not yet give us enough genomic data to draw reliable conclusions about the relatedness among Cyp. parviflorum individuals. Of the samples tested, many of the sequences produced with the same primer sets were almost identical. Primer sets that did not produce consistent results will be re-tested and additional primer sets and samples will be tested to extend this study and assess diversity among plants in different areas.

My Story

I got to know about the New Hampshire Academy of Science through a friend in 2021 and thought it would be a cool experience to try out. I quickly applied and signed up for the three-week summer camp for scientific research. I chose to research the genetic diversity of the Lady’s Slipper Orchids which was a less commonly researched topic in the scientific community. A few days into the first week, I was conducting my research with confidence and always excited to come back to the lab.

Due to the pandemic, the camp had to be in a hybrid format which meant I worked at home for a few days a week. On those days, I packed my DNA samples and other various liquids in a freezer box and brought them home to continue the experiments. Although there were no supervisors to help me, I followed my lab notes of the procedures to perform the experiments consistently. Even so, the results I obtained at home were similar to the ones I received in the lab.

At the end of the program, I worked with my mentor and many other students working on similar projects to share my findings. I wrote a paper about my research which was sent for review for the AJAS conference. I am looking forward to all the events of this year's conference!

Gel electrophoresis

Of all ten primer sets tested, 6 had visible bands. The six are M130, M136, M139, M142, M681, and M880. M370 was sent for sequencing but we later realized that there were double bands present, which did not show usable results.