Going with the flow: a new and improved milkweed project, finally getting results

If there’s one thing that I’ve learned from my three years of research, it’s that its super important to go with the flow. Since my last update my milkweed project has taken on an entirely new shape. After discovering that glyphosate herbicides do not act as a proxy for connectedness, I decided to use a different approach and expand on my project from last summer.

My project last summer focused on quantifying genetic diversity of milkweed and creating a coefficient of relatedness across 14 transects. Functional trait data such as plant height, stem width, and leaf length were collected in 4 locations as well as spatial data and leaf samples. I worked on extracting all samples and doing PCR on them. Unfortunately due to issues with primers and equipment issues it was difficult to finish this project.

This summer, with the help of two other undergraduates, and two graduate students, this project has taken a whole new form correlating functional trait data to clonality and spatial layout. We unfortunately had to throw away all of the extraction and PCR I had completed last summer due to decay over time. Thankfully our original leaf samples and data collected in the field were preserved so the collection process did not have to reoccur.

Luckily this summer we have been able to do the project all over again with many more hands and much more knowledge. We have again reduced the number of transects down to just 8 of the original 14, and with around 200 individuals subsampled of an approximate 400.

The DNA extraction process from the leaf is complete, though a few redos may need to be done in the future. Afterwards PCR was used. Polymerase chain reaction amplifies, and then copies segments of DNA to make analysis easier. The protocol includes using both a forward and reverse primer. Additionally a TAQ solution catalyzes the polymerase. Many of these reactions can only occur at specific temperatures. Because of this, a thermocycler is used to cycle through the annealing temp, denaturization of the DNA, and elongation. Each individual has undergone PCR at least once, though errors necessitate some redos as well as needing duplicates.

By having duplicates of PCR we can show our error rate which is important to understand whether our study is scientifically valid.

Last summer, and through the semester most of our data and results did not look good. This meant we had to continue to improve our protocol and continue redoing things. Finally, our plates and data have started to look good. We use a genotyping program called Geneious to analyze the results of our PCR. This software is very expensive so we do not own it. Though we are able to use another professor’s copy, sharing the computer between multiple labs, and many different student’s and professor’s needs has proven difficult. We luckily were able to get a free trial for a few weeks which helped to alleviate the most pressing portion of the genotyping.

Now that we have “finished” genotyping, we are aware of which ones we need to redo, and have begun to do analysis on our results. I will talk more about our analysis process in my next blogpost.