Analysis of Milkweed Genotyping

With just a few individuals left to send off as redos, most of the focus on the milkweed project has moved towards analysis. Analysis is inherently tricky because there is a lot of trial and error. There are essentially a few options for analysis. You can use someone else’s software, you can modify someone else’s to fit your needs, OR you can make your own. The last one is obviously extremely time intensive, so our hope was to find a software we could use to fit our needs.

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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.

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Milkweed Connectedness Update 1:

My original plan was to use common Milkweed as a study system to understand the impact of clonality and group survival. By intentionally adding a pathogen, such as an herbicide, the spread of the negative effects can be witnessed in a clonally connected plant. The goal of this experiment is to see how far the pathogens travel in a patch, how long it takes for other plants to die, and if there is any preferential sharing. For instance, sometimes younger plants are favored in sharing. Herbicide will be used as a proxy for connectedness and physiological integration. 

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Microsatellite Genotyping of Milkweed:

Milkweed and monarchs populations have been steadily declining due to habitat changes and a whole host of other factors for many years. Conservation efforts include adding more diversity to milkweed populations as lower diversity of milkweed is dangerous because it leads to more susceptibility for milkweed extinction. Analyzing the diversity of milkweed is imperative for replanting efforts and conservation goals of both the monarch and milkweed as milkweed is the most important plant for larval monarchs. My goal for this experiment is to evaluate patches of milkweed and quantify genetic diversity so as to help conservation efforts. I will sample and map the stems of common milkweed in five populations that have been sampled for the past four years to discover how diverse these populations are by using microsatellite markers.  Sampling will occur in 5 populations during the month of June, and the distance between the different plants, ramets, will be recorded on site. Once back in the lab, DNA extraction will occur using a specific protocol. After extraction, Polymerase Chain Reaction (PCR) will be done on the DNA to amplify sections of the DNA sequence using 8 primers developed for this specific species of milkweed, Asclepias syriaca. PCR is done using a MyTaq kit and a thermocline machine. The sections of the DNA amplified are microsatellites in this case. Microsatellites are repeated sections of DNA unique to each plant. However, because milkweed is very clonal, or makes clones of itself, many plants in a patch could be genetically identical, or having the same microsatellite sequence, which means there is very low genetic diversity. Once these sections are amplified, fragment analysis can occur so as to compare the length of microsatellites to evaluate the clonality of milkweed. Additionally with use of the ramet density data, the probability of any plants being related can hopefully be correlated to their distance.