The goal for this summer was to have sampling, DNA extraction, Polymerase Chain Reaction (PCR), and fragment analysis sent off (to be later analyzed) completed on all 400 of the subsamples. Sampling and DNA extraction were finished. PCR unfortunately was not, delaying fragment analysis as well. Though the PCR protocol was developed during spring semester of 2017, new primers and a host of other problems made this step tricky. The first batch sent off for analysis came back blank, requiring adjusting of the protocol and the second had nebulous results. A delay in shipment meant that only 3 of the 7 primers were available for much of the summer. However, all extractions were completed successfully, and in the last remaining weeks of July, PCR was done on all 400 of the plants for 3 of the primers. All PCR products were shipped off for fragment analysis. Training began on using a fragment analysis software, so once results do come back, analysis will be smoother.

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Labwork (PCR)

Following DNA extractions, it was time for amplification of DNA through Polymerase Chain Reaction (PCR). PCR amplifies the DNA by denaturing, copying, and synthesizing over and over again. Denaturing occurs via temperature, first during the DNA extraction process, and then again using the Thermocline machine (or PCR machine). The thermocline helps to regulate temperature allowing for PCR to go through its different steps, such as synthesis or denaturing. Synthesis occurs using a special polymerase Taq.

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Following the collection of data and samples, it was time for lab work to begin. The first step of my procedure was DNA extractions. With around 800 plant samples, I quickly realized financially and time wise doing all 800 would not be practical. This led to many discussions on how to subsample. Was it better to do more transects, with fewer plants from each transect? More plants, with fewer coverage of overall transects? How do you account for the difference in densities between transects? Overtime, with more and more discussions, it became clear that sampling more transects would be a better option, even if that meant fewer plants per transect. Additionally, for any transect with ~30 or fewer plants, the entire transect would be sampled. For any plant over that, a subsample would be done using a random number generator to randomly select which plants should be extracted.

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The month of June was predominantly spent on fieldwork and preparation for fieldwork. Preparation included obtaining materials, packing and planning ahead for the visits. There are 5 sites for field work: Presquile National Wildlife Reserve (PWR), Blandy Meadows (BLD-M), Blandy Thistle Thicket (BLD-T), Sky Meadows (SKY), and Greenspring (GRN). For PWR, BLD-M, BLD-T, and SKY, overnight visits were required. We were fortunate enough to have access to research housing for all of these. PWR is an island so that required intensive planning as nothing could be forgotten. Once we arrived at each site, we jumped right into fieldwork. [Read more…]

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.