MonthOctober 2019

DNA Extraction from Mimulus guttatus leaves

In order to perform a DNA extraction from M. guttatus leaves, we began by labeling three 2.0 mL tubes with the sample codes GS1, GS2, GS3. In each tube, three stainless steel beads and a small amount of leaf tissue were placed and attached to the modified reciprocating saw rack to be shaken. The tubes were then spun down in the centrifuge to pull down plant dust. We then added 434 microliters of preheated grind buffer to each tube and incubated it at 65 degrees celsius for 10 minutes in a water bath. We then added 130 microliters of 3M pH 4.7 potassium acetate and inverted the tubes several times and incubated the tubes on ice for 5 minutes. The tubes were once again centrifuged for 20 minutes at maximum power.

Three new sterile 1.5 mL tubes with the sample ID that it was assigned when it was collected were labeled and filled with the supernatant. We then added 1.5 volumes of binding buffer, and 650 microliters of this mixture transferred to Epoch spin column tubes. The column tubes were centrifuged for 10 minutes and the flow through was discarded. The flow centrifuge and flow through steps were completed until the mixture had all gone through. A dry centrifuge with the spin column tubes was then done for 5 minutes. The collection tubes were discarded and and the columns were placed in labeled sterile 1.5 mL microcentrifuge tubes. 100 microliters of preheated pure sterile water to each tube and was left alone to stand for five minutes and then centrifuged for 2 minutes to elute the DNA.

Using Geneious Software to compare animal tissue DNA extractions

In this lab, we were able to download a program, Geneious, which allowed us to access the sequences produced by our DNA extractions of animal tissue from sushi. From the 4 samples of DNA, only 2, GS01 and GS02, were viable to produce sequences from both the forward and backward primers. Starting with GS01, the forward primer sequence had an HQ (High Quality) of 74.2% and the reverse primer sequence had an HQ of 25.7%.Using the DeNovo feature, we were able to combine the forward and reverse primer sequences and edit the sequence to produce a consensus sequence with an HQ of  81.9%. This number was produced only after editing sequences, which consisted of cutting off the ends of the sequence and erasing or fixing single bases. GS02 was subjected to the same DeNovo feature and editing process to produce a consensus sequence with an HQ of 78.8%.

We then were able to BLAST (Basic Local Assignment Search Tool) each of our consensus sequences, which allowed us to search a database for highly similar sequences. We were able to determine how well the sequences matched based on the Grade of the sequence. We selected 5 species to compare the consensus sequences to. Using this tool, I was able to conclude that the sample labeled GS01 was most likely Thunnus Albacares, which is a yellowfin tuna meaning the sushi market was in fact selling tuna. Sample GS02 was also analyzed using the BLAST tool and after comparing the consensus sequence it was determined to most likely be Seriola quinqueradiata, which is a yellowtail fish as advertised in the sushi market.

While the sequences were very similar, there were polymorphisms. For GS01, there were 4 polymorphisms which resulted from editing since some bases were erased due to convoluted fluorescence. There were also may bases that were not definitively determined, thus the were discrepancies in those as well. Base 511 had GS02 consensus sequence of a T nucleotide, however, there the rest of the species had a consensus in which there was no base there. There was also a similar situation at base 498. At base 121, the fluorescence was confusing and caused a polymorphism which is depicted in figure 2.


Figure 1: This is a GS01 polymorphisms in which a T nucleotide was incorrect.

Figure 2: GS01 had a consensus sequence that produced a T nucleotide, however the species consensus was a C.

When GS02 was compared to 5 other sequences, there were 22 polymorphisms. Although there were many polymorphisms, this was mostly due to including a 2 species that were not as similar to the sequence that were included in the comparison. However, when GS02 was compared to just the Seriola quinqueradiata, there were no polymorphisms. An example can be seen in Figure 3.

Figure 3: A GS02 polymorphism when comparing 5 other species.

Field Trip 9/23/19

A Mimulus guttatus by a water fountain and exposed to large amounts of sunlight

On our field trip, we traveled to Muir Beach, near Mountain Tamalpais. On this trip, we were able to see multiple populations of Mimulus guttatus in different environments. Due to the different environmental pressures, these populations demonstrated different traits that correlated to adaptive pressures.

These mimulus are not yet matured, but will most likely flower due to their environment

For example, the first mimulus plants we saw were exposed to high amounts of sunlight and had constant water supply from a fountain. This caused there to be flourishing populations that were also flowering, thus signaling a readiness of maturity and to pass on genes to next generations. The plants in this area were healthy enough and in a good position, so they flowered, and bees most likely acted as pollinators for these plants since the provide an ideal landing platform for bees and red spots on the petals that could be attracting the bees. We spoke of the likeliness that a pollinator would travel far to spread the mimulus’ pollen, and determined that the mimulus guttatus that were closer would be receiving those genes rather than further ones. This would mean that there is a high probability of inbreeding, which can be dangerous to the gene pool. We were also able to see what mimulus looks like in its primary stages of life in this area as another population began to flourish. It is also worth mentioning that the mimulus populations were usually found around horsetail and ferns.

The mimulus here comes from a flowering plants and demonstrates the ideal bee landing petals and red spots

We then traveled to a more shaded part of the terrain where a creek was found. After traveling up the creek, a population of mimulus guttatus was found, but the populations was not flowering. The reason for the lack of flowering can be explained through the location of the plants. They were located in a shaded creek area that would get flooded in a few months time and had very little pollinator traffic, thus, along with shortening of the days, the mimulus reacted to these cues by not reproducing. These populations are called sink populations and they will not be participating in breeding with any other populations.

These mimulus are not flowering because of the lack of reproductive cues

These different populations are most likely not to be very connected, but it all depends on the distance that pollinators travel and the seed dispersal that occurs. Mimulus guttatus is a very diverse plant and continues to demonstrate the adaptive capabilities it possesses.

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