For Lab 8, I was preparing DNA extractions from Mimulus guttatus leaf samples. I had three samples: CATB JK 01, simplified to JK01, SHOR 002 AJC, simplified to JK02, and TROJ 003 AJC, simplified to JK03. Although broadly similar to the prior fish DNA extraction, plant tissue is more dense, so we first had to pulverize it using steel ball bearings for 40 seconds using a modified reciprocating saw to masticate the tissue. Following mastication, the tubes were spun down to concentrate the tissue powder, and had 434 microliters of warm grind buffer added to each tube. The grindate was then incubated for 10 min in a water bath, after which 130 mL of potassium acetate was added. The tubes were then incubated on ice for 5 minutes to precipitate out DNA. Next, the sample was vortexed at 15000 RPM in a microcentrifuge for 20 minutes to separate the supernatant from the solid leaf tissue particles. Next, the supernatant was transferred to new 1.5 mL sterile tubes labelled with the sample IDs ( JK01, JK02, and JK03). After that, I added 600 microliters of binding buffer to bind the DNA to a flow-through collection column. I then passed the buffer solution containing the DNA through an Epoch spin column for 2 cycles of concentration in the column followed by two washes with 500 mL 70% EtOH, with a final 5 min spin cycle to remove the remaining EtOH. After this, the columns were moved to the new, labeled 1.5 mL microcentrifuge tubes, and had 100 microliters of sterile H20 added to collect the eluted DNA. After this, they were spun for a final centrifuge cycle for 2 minutes for elution, then taken away by Dr. Paul and stored on ice for later processing.
For Lab 7, I practiced using phylogenetic tree analysis methods on 10 of the DNA sequences from my DNA isolation project earlier this semester. First, I ran the Jmodeltest program on my assembled BLASt hits in order to determine the best methods for analysis; the AIC gave HKY as the best molecular model, whereas BIC turned up H80. Next, I made a series of phylogenetic trees using different programs to test them out. First, I made a MrBayes tree using the HKY85 model. This gave back an identity suggestings that Red Drum (Sciaenops ocellatus) was the most likely ID for my fish. Next, I did a Maximum likelihood tests with RAxML Bootstrapping, followed by one with PHYML bootstrapping. Both gave similar results as MrBayes, but with less support of side trees. Both gave weak posterior distributions, likely due to insufficient sample sizes.
I assembled the forward and reverse reads results for the JK-02 and PR-03 gene assemblies after downloading the info off of Geneious. Unfortunately, the read quality was too low to be able to assemble the reverse and forward reads of the JK-02 sequences, both of which had about 20 polymorphic sites each close to the ends. It was an interesting discrepancy; the read quality was low, but polymorphic sites were also low. Despite the low read quality, the DNA was successfully blasted and indicated that the fish was in fact the Red Drum, Sciaenops ocellatus. The DNA samples from PR-03 were much better in quality overall and assembled easily without any polymorphic DNA sites. The species ID was not as certain, however, though it was confined to only very closely related species in the genus Thunnus, namely T. alalalunga and T. orientalis. I was informed that the samplw was supposed to be tuna, but not which species, so I am unsure of whether or not the marked species was correct for that individual.
On Sept. 24th 2019, me and my Molecular Ecology class took a field trip to the coastal areas in the vicinity of Mt. Tamalpais state park. Due to a navigation error, the SUV I was driving took a wrong turn, so we missed some of the initial field trip time. Eventually however, we met up with the main group at a spring by the side of the highway near Stinson Beach. Here, Dr. Paul introduced us to a perennial form population of Mimulus guttatus in order to show us the morphological forms the species can take when exposed to constant moisture, rather than the deceased annual populations we had seen before. After this, he took us to a creek near the Muir Beach trail to show us another population that was on a different annual cycle than the ones one the serpentine barrens. As they grew on a dry creek bed during the summer season and the creek region was less dry in the summer, this population was summer-annual rather than winter-annual. Thus, by observing and sampling them, we get to see yet another form of Mimulus guttatus expression, and add more to our gene library. I also got to see a lot of native fish and crayfish in the creek, which was interesting. Overall, I got to see more new forms of Mimulus than I had seen before, which helped my better grasp the full variation of the species.