A Real-Life Case of Stump the Forester

One of the best aspects of working in the tree care industry is the collegial and collaborative mindset shared by its experts and practitioners. If I have a question, I can send it to my peers in academia or industry, and they are quick to offer any insights they have. Recently, I received an email from Charlie Marcus at Legacy Arborist Services with a similar request. A tree had been removed without the required permit, and the species identification was in question—an important detail in determining the level of protection the tree should have received and the consequences of its unauthorized removal.

Figure 1. The stump that had us stumped (Photo Credit Charlie Marcus)

As is common in many Florida communities, live oaks (Quercus virginiana) are often the focus of local tree protections. Had we been in Wisconsin—where I first learned dendrology—one telltale characteristic of oaks would have been their ring pattern. Many species, such as white oak (Quercus alba), are ring-porous, featuring large vessels in the earlywood and smaller vessels in the latewood. Live oaks, however, are not considered truly ring-porous. While they exhibit some ring structure, they are more accurately categorized as semi-ring-porous or even diffuse-porous.

Armed with only a few photos of an aged stump and this newfound knowledge, Charlie had managed to stump this forester. (I was torn between labeling this moment a TREE-SI or paying homage to Joe Anderson’s long-running column in the Florida Urban Forestry Council Newsletter—and ultimately decided, “Why choose?”)

Fortunately, I happen to know a local molecular biologist who had recently mentioned her efforts to teach genetic barcoding to her students. Dr. Sarah Wilson, a professor at the University of Tampa, teaches biology, cell biology, and genetics. As part of a recent project, she had her students collect samples of unknown plant species, extract DNA from the specimens, and compare the sequences to an online database in order to make accurate identifications.

I texted her to see if the same methodology could be used on our mystery stump, and she was glad to give it a try. Even better, I convinced her to co-write this blog post to explain what genetic barcoding is—and to walk us through the multi-step lab procedure we followed. It was a great learning experience for me and I hope to share it with you in this post.

What is DNA Barcoding? (by Dr. Sarah Wilson)

DNA barcoding is a method used for identification of the genus and species of organisms when traditional taxonomic methods aren’t possible. DNA barcoding requires a very small amount of tissue (the size of a grain of rice). The tissue sample can be taken from a leaf, flower, fruit, roots, trunk or branches. DNA is extracted from the tissue sample and a short segment of a specific gene or genes is analyzed. In plants, a segment of the gene that codes for the RuBiCO enzyme is used. RuBisCO is a critical enzyme involved in the process of carbon fixation during photosynthesis and is found in the chloroplast genome of all plants. This short sequence of DNA is called a DNA barcode and works like the UPC (universal product code) on a product at the grocery store. Each item at a store has a unique UPC so that the cash register knows which product to charge you for. In DNA barcoding, a short segment of about 500 nucleotides (the building blocks of DNA) is unique for each species. Once the sequence of nucleotides (A, T, G, Cs) in the RuBisCO gene is determined, it is compared to known sequences that have been deposited in databases and the identity of the sample can be determined.

Figure 2. Dr. Sarah Wilson in her natural habitat, the genetics teaching laboratory at the University of Tampa.

Stump Identification in 20 Easy Steps

As indicated by the bag, the chips to the left came from Charlie Marcus’s mystery stump. The sample was hand delivered to Andrew by Charlie at Trees Florida 2025 in Palm Coast (a great event for arborists, urban foresters, and their families).

Wood is a mix of living (e.g., parenchyma) and dead cells (e.g., xylem). Although Sarah typically has her students collect samples from live foliage for DNA extractions, we were cautiously optimistic that we could obtain usable material from the stump samples.

We took small shavings—each about the size of a grain of rice—from four different locations, starting just behind the bark and moving inward. We were careful to avoid including moss from the outer bark in our samples (we’ll find out how successful we were when the results come in). Given the age of the stump, Andrew was concerned it might be contaminated with DNA from decay fungi, but Sarah explained that the primers they were using were specifically selective for plant DNA. Primers are short segments of DNA that are used in the barcoding process in plants to target the RuBisCO gene. Fungi do not have the RuBisCO gene in their genome, so they would not interfere.

Figure 5. Small sample with lots of data

A tiny sample in a microcentrifuge tube. Only about 10 milligrams (about the size of a grain of rice) is required for the DNA extraction step. We set up four of these sample tubes with approximately the same amount of tissue in each.

Figure 6. Plastic pestles (not to be confused with plastic pesto)

We used these little plastic pestles to grind up the sample in a lysis buffer solution. Lysis means to disintegrate cells by breaking cell walls. The buffer is a liquid containing detergent that helps release the contents of the cells, and ultimately the DNA.

Andrew’s sample of bark tissue after grinding up the sample using the tiny pestle. We repeated this for 4 samples.

After breaking up the tissue, samples were heated at 65℃ for 10 minutes. The heating step helps separate proteins in the sample so the DNA can be purified. Note the random placement of the sample tubes in the heat block, thanks to Andrew.

Next, samples were placed in a balanced configuration in a mini centrifuge and spun for 1 minute at 13,000 rpm (rotations per minute). After centrifugation, the large pieces of solid material form a “pellet” at the bottom of the tube and the DNA remains in the liquid layer that forms above the pellet. Sarah insists that having the hinge for the cap facing outward is the only way to place the tubes in the centrifuge.

Back to the heat block. A silica resin was added to each of the samples. Silica is a positively charged material and DNA is negatively charged. Opposites attract and the DNA sticks to the silica. Heating the samples at 57℃ in the heat block helps with this process. Andrew still thinks it is funny to place the tubes in random locations in the heat block. Sarah disagrees.

The silica/DNA mixture was washed several times to remove any contaminants (like protein or other cell parts). We used a vortex to mix the resin with the wash buffer. A vortex is a fancy piece of lab equipment used for mixing samples. After two rounds of washing, the DNA was removed from the silica resin using pure water.

The samples were tested using a Nanodrop to see if any DNA was purified after all of the steps completed above. We were cautiously optimistic, but whenever you are working with old samples or dead material, there is a chance that the DNA could be broken down. The nanodrop is a little machine that measures how much DNA is present in your sample. We put 1 microliter (= 1 millionth of a liter) from each sample onto the nanodrop to get a reading.

The results for one of the DNA samples showed that we purified 31.5 ng/µl (nanograms per microliter) of DNA. This is a tiny amount of DNA, think of it as about 31.5 ppm. Three out of four samples worked and contained enough DNA to be detected by the nanodrop and these three samples (numbered 1, 2, and 4) were used in the later steps.

The three samples (1, 2, 4) that contained DNA were used to set up PCR reactions. PCR stands for Polymerase Chain Reaction, and is the next step in the DNA barcoding process. Primers for the RuBisCO gene were combined with enzymes and the purified DNA to make billions of copies of the barcode region. Once the PCR reagents were combined, they were spun in a mini centrifuge before the next step. Over an hour into the process, Sarah didn’t feel the need to balance the centrifuge—despite knowing photographs would be taken.

The PCRs were set up for 3 DNA samples and 1 negative control. The negative control has all of the ingredients for a PCR, but no DNA was added. The negative control was included to make sure there was no contamination. The numbers on these tubes were written by Andrew (although one might easily believe they were scribbled by a raccoon).

The samples were placed in a thermocycler to run the PCR protocol. A thermocycler is a programmable heat block that cycles between different temperatures for specific lengths of time. The PCR conditions depend on the primers that are used, so this is a specific protocol for plant barcoding. The temperature cycling allows for the RuBisCO gene to be copied billions of times. DNA is so small that billions of copies are needed in order to see it in the next step.

After about an hour and a half in the thermocycler, the PCR reaction was complete and the samples looked exactly the same as they did when they went into the machine. The magic was all happening at the molecular level, just trust the process.

The next step was to run an agarose gel in order to see if the PCR worked. Agarose is a carbohydrate (sugar) purified from seaweed and it is used to make gels for DNA analysis. The process is similar to making old fashioned gelatin on your stovetop. Agarose is like the gelatin powder and a specific amount is weighed using a balance.

Figure 19. The solution was right in front of us.

The agarose is combined with a solution containing ions. The TBE (tris, boric acid, EDTA) solution is like the water that is added when making gelatin and a specific volume is measured using a graduated cylinder.

Figure 20. Microwaves…they can do more than cook ramen.

The agarose and buffer solution were combined in a flask. We heated the agar in a microwave, much like you would when making gelatin. The solution started out cloudy and the grains of agarose were visible. After the boiling step, the agarose dissolved into the TBE solution making a clear (HOT!) liquid gel.

The agarose gel was poured into a square mold that includes a comb-like insert. When removed, the comb creates small rectangular wells for holding DNA samples. We then let the agar cool and form a semi-solid gel for electrophoresis.

A portion of each sample was carefully added to the wells of the gel using a micropipette. A ladder sample was also added to the gel and contains DNA samples of known sizes (nucleotides), so that we can compare our PCR samples to see if they are the expected size. Electric current was applied to the gel (120 volts) to allow the DNA to migrate through the gel. The DNA moves through the gel material based on size. Longer pieces of DNA move slowly through the gel and smaller pieces of DNA move faster through the gel. We let it run for about 20 minutes to allow adequate separation of the ladder.

The resulting gel image showed that all three PCR reactions worked! The first two lanes of the gel contain the ladder. The first was Sarah’s demo and the second ladder was expertly loaded by Andrew (it is tricky!). The samples in the next lanes show that DNA samples (1, 2, and 4) were amplified by PCR. Basically, billions of copies of the RuBisCO gene were made using the DNA that was prepared in the first steps. The last sample is the negative control and does not have any result, which is a good thing indicating that there wasn’t any contamination.

Figure 24. Getting ready to ship out for futher testing

The final samples! Three of the four original DNA extractions contained DNA, as determined using the nanodrop. All three of those samples showed that the PCR worked when we ran a portion of the samples out using gel electrophoresis. The remaining volume of each sample will be used to sequence the barcode.

To be continued…

The samples have been sent off to Genewiz for sequencing analysis. When we receive the report, we’ll have the barcode information needed to search the database and identify the species. When we do, we will share our results in a future blog post. Hopefully, we don’t end up with an identification of the moss growing on the bark—or something like Sitka spruce (Picea sitchensis), which is native to Alaska, not Florida. If we do, Andrew will get the full PCR experience: pipette, cry, repeat.

About this blog

Rooted in Tree Research is a joint effort by Andrew Koeser and Alyssa Vinson. Andrew is a Research and Extension Professor at the University of Florida Gulf Coast Research and Education Center near Tampa, Florida. Alyssa Vinson is the Urban Forestry Extension Specialist for Hillsborough County, Florida.

The mission of this blog is to highlight new, exciting, and overlooked research findings (tagged Tree Research Journal Club) while also examining many arboricultural and horticultural “truths” that have never been empirically studied—until now (tagged Show Us the Data!).

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