Warren Chatwin and Dr. Leigh Johnson, Department of Biology

My project “Genetic Diversity and Conservation of Old-Growth Cross Timbers Forest” has been undoubtedly the best learning experience of my undergraduate career. The project did not go at all like I had planned, but it gave me a taste of realworld scientific exploration and allowed me to practice some of the things I’ve learned in class. I’ve adapted to challenges, changed my plans, overcome obstacles through creative thinking and sheer diligence, and collaborated with colleagues who are working on the same organism. These skills will be invaluable in my future career as a research professor. I will see this project through no matter what road blocks are still ahead, and I hope to wrap things up by mid-summer at the latest.

I set out to sample Post Oak (Quercus stellata) leaves across the range of the Cross Timbers Forest (Central Kansas to Northern Texas) to measure the genetic diversity in the forest, which has been reduced to 1.6% of its original range. I had planned to collect newly-emerged leaves because soft, young leaves haven’t built up the phenolic compounds that can interfere with DNA extraction. However, due to an early spring in the Midwest and a long finals week here at BYU, I left Provo two weeks after the leaves had emerged. When I arrived in Oklahoma, to my dismay the trees had suffered a caterpillar outbreak. Some bare trees had started putting out a second flush (set) of leaves. Recalling research I’d seen in my plant physiology class at BYU, I didn’t collect from those trees since the problematic phenolic compounds, which are a tree’s natural chemical defenses, would abound in the second-flush leaves. I had to search hard for the youngest, most un-chewed leaves, which even then were sure to contain phenolic compounds. The most frustrating part of this outbreak was its specificity: the caterpillars preferred Post Oaks over any other surrounding tree (even other oaks!). The outbreak also covered a wide area, stretching from Kansas all the way to Southern Oklahoma. These complications slowed my journey and increased the chance that the leaves wouldn’t keep well or process well. I had followed the advice of Dr. Andrew Hipp, a fellow scientist studying oaks in Chicago, about the best way to keep the leaves fresh between collection in the field and processing in the lab. During the three weeks I spent on the road sampling, I experimented with different coolers, ice shapes, and storage strategies to keep the leaves from wilting or getting wet in ice melt, and sometimes it wasn’t easy. Even with all of these obstacles, I was able to gather 162 samples from 23 sites, place them in individual plastic bags, and obtain GPS coordinates for each sample. I returned to Provo, placed in the -80°C freezer for storage, and put the project on hold for the summer (due to a previous commitment).

I returned to BYU in late August and began to extract DNA from the leaves, using Dr. Johnson’s phenol/chloroform based protocol. However, after continued research and communication with Dr. Hipp in Chicago I decided that the approach I had proposed – to determine genetic diversity by looking in highly variable regions of the chloroplast genome – was not the best approach to use in this case because oaks hybridize so freely that the chloroplast line may not be specific enough. Dr. Johnson and I decided to try a different approach using nuclear Single Sequence Repeats (nSSR), and collaborated with Dr. Maughan in the PWS department, who has more experience in that approach. We planned to use primers generated from a closely-related oak species, Quercus alba, whose genome (unlike that of the Post Oak) was already partially assembled, and whose nSSRs were already developed. Dr. Maughan taught me how to select a good mix of nSSRs of varying repeat sizes, and we ordered primers for them. We then tested these primers on DNA extracted from my samples and found that some primers successfully amplified the DNA, but few were truly polymorphic (showing up with different repeat lengths in different individuals). While screening for these nSSRs we also did a genomic reduction and partial 454 sequencing run to generate sequence data for Post Oak. We then isolated nSSRs from different Post Oak samples (because they would have more polymorphic nSSRs) and ordered primers based on those newly isolated nSSRs.

Meanwhile, things were further complicated by the difficulty of extracting clean DNA from samples laden with troublesome phenolic compounds. I switched to Dr. Maughan’s phenol/chloroform protocol and still couldn’t get clean DNA, so I went back to the literature to see what I could find. A paper published just last year outlined another method for oak DNA extractions (performed on a European oak species). We tried adopting that protocol (using the Quiagen DNeasy Plant Mini kit + Polyvinylpyrrolidone), and still weren’t having success removing all the phenol contaminants. At this point, with some nudging from Dr. Maughan, I did something that terrified me a little bit: I sent an email to the corresponding author of the paper above
(who works in Spain) and to all the professors I knew were working on oaks. Most responded quickly with helpful comments, but overall it was Tim McLeary, a lab manager at Notre Dame, who helped me the most. He listened as I explained what I’d done so far and suggested things I might try to get better results, from adapting future sampling methods to changing the DNA extraction protocol to adjusting the PCR set-up. We spoke just yesterday, so this is where my project currently stands.

My goals now are to implement these new suggestions and, if I get clean DNA that works consistently in PCR (it’s been sporadic), to move on to the next phase of the project. I will use a technique called “M13 tailing” to ‘attach’ a specific sequence to each PCR-amplified product. I will then be able to use that sequence to isolate and sequence the associated nSSR and quantify the exact amount of nucleotide differences between nSSRs. The more differences there are across the sample range, the healthier the Cross Timbers forest is. I will use the nSSR data to make recommendations for conserving the tracts of land that exhibit the highest diversity to help preserve this littlestudied species. Perhaps oaks are under-studied because working with oaks is inherently difficult, but perhaps it just needs more researchers to take interest. This
project has ignited my passion for research. Even with the difficulties – or perhaps because of them – I have learned far more by getting out and doing my own work than I could have by sitting passively in the classroom. I’ve felt frustrated and exhilarated at different times during this project, I’ve learned to ask for help from colleagues, and I really got my hands dirty searching for real results. I know these skills and lessons will prove invaluable to me throughout my life, especially as I continue my education and prepare to contribute to the academic world.