Qualitative and quantitative assessment of DNA extracted from alternative tissue samples of woody plant species Kirthika Ramakrishnan Mentored by Tami J. Imbierowicz and Jaclyn A. Madden Methods and Materials Fresh live leaf samples were clipped from the branches of sample species. Dead leaves were collected from the base of the tree and cambium was extracted using a bore. The bore was inserted at least 2 inches into the tree, to make sure to get past the dead bark tissue layering the trunk. Condition, location and identity of the organism that the sample was collected from was recorded. All samples were labeled with a sample number and stored at -20 °C. DNA was extracted using methods and materials outlined in the “Using DNA Barcodes to Identify and Classify Living Things Manual and Kit,” produced by Carolina Biological. Unamplified DNA was analyzed using the UV/Vis spectrophotometer (UV/Vis spec) at 260 nm, 280 nm, and 320 nm to assess the quality and quantity of DNA extracted from each sample. Once the DNA extract was amplified with PCR, it was run through Gel Electrophoresis and sent to Genewiz® for sequencing. Sequences were run through the National Center for Biotechnology Information (NCBI) database to test if the sequences of the extracted samples could be used to identify the organism from which it was extracted and identify the gene as the rbcl gene. Introduction Case studies requiring the use of woody plant species often have protocol issues due to limited access of the appropriate samples. The standard source of DNA for a woody plant species is a fresh, live leaf from the canopy of a tree. The live leaf is hard to access and is only available for a short period of time compared to the timeline that a study usually spans. The height of many trees makes samples hard to access, let alone without contamination. In this is study, alternative tissue samples from Platanus occidentalis L (American Sycamore), Quercus macrocarpa (Mossy Cup Oak), and Carpinus caroliniana (American Hornbeam) were examined for their genetic information. Tissue samples of dead leaves, live leaves and cambium(cellular tissue past the dead bark of a tree) were collected. The purpose of doing so was to see if that when compared side by side can the dead leaf or the cambium be used as a stand in for the live leaf. Some studies have indicated that the commonality between cambium and live leaves is higher than any other tissue sample tested (Fay, Periziosi & Ztynska, 2012). The RuBisCo (rbcl) gene can be found in all parts of a tree, even within the cambium. So in this study, the rbcl gene was used as a common factor to compare genetic information extracted from the cambium and the dead leaves to the live leaves. This was done to see if either the cambium or the dead leaves can be used as alternatives to the live leaves in a genetic study. Results Figure 1: The sequence depicted in A is the sequence derived from the live leaf sample of the Mossy Cup Oak. This sequence is very similar to the one depicted in B, which is the sequence from the cambium of the Mossy Cup Oak. In both of these sequences nucleotide peaks can easily be identified and the sequence is long enough to determine the original organism and the rbcl gene that was extracted. In sequence C, the sequence from the dead leaf sample, very few nucleotides can be identified and the sample cannot be matched with the organism or the rbcl gene. Graph 1: Shows the box plot of the length of sequences of the samples. Sequences at the higher range show a high percentage of the gene the was usable. A chi-squared test comparing the live leaf to the cambium gives a p-value of well above one, which means that any difference in the data is not random change. While the test comparing live leaves to the cambium gave a p-value of only 0.04. This p-value indicates there is no statistical difference in between the samples DNA Sequences of Mossy Cup Oak C T T G G C A G C G T T C C G A G T A A C T C C T C A A C C T G G A G T T C C G G C C G A A G A A G A T A T C T T G G C A G C C T T C C G A G T A A C T C C T C A A C C T G G A G T B N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N G N N G N N G N N C Results (continued) All samples analyzed with the UV/Vis spec were shown to be pure enough for genetic analysis. The differences in concentrations, determined by the UV/Vis spec, by a chi-squared test only had a p-value of 0.03. This value means that the difference in concentrations were caused by random chance not an underlying variable. When the sequences of the cambium and live leaf samples were run through NCBI, they could be used to identify both the organism and the rbcl gene that was isolated. Figures 1a and 1b reflect such sequences. The sequences that were derived from the dead leaf samples were short and often gave results that did not match the gene or organism that sample were taken from, and some gave no results at all. Such as the sequence in Figure 1c. Refer to Graph 1 for statistics on the extracted DNA sequences. The dead leaf compared to the live leaf or cambium samples had a wide range when it came to the number of base pairs that were readable. Some of the samples could give results that exactly match those of the live leaves and some could have no results at all. References Fay, M.F., Periziosi, R.F. & Ztynska, S.E. (2012). Comparing the use of leaf and cambium tissue in a single study of tropical trees. Tree Genetics and Genome, 8, 431-437. DNA Learning Center, Cold Spring Laboratory. (2014). Using DNA Barcodes to Identify and Classify Living Things. New York, Carolina Biological. Conclusions The study’s purpose was to see if an alternative tissue sample can be used in place of the live leaf in a genetic analysis. The lack of a statistical difference in the UV/Vis spec results supports the hypothesis that the dead leaves or the cambium can be used instead of the live leaves. However, the wide range of the dead leaf sequences make them an unreliable source of data. The results obtained from the cambium can be used in tandem with the live leaves for any purpose that requires finding genetic information from the organism. Even so, the cambium of a tree can easily be stored for an extended period of time and can be extracted from the organism at any point in the year, giving easy access to data at any point in the study. This suggests that not only can cambium be used instead of live leaves; it might be the better choice. There is the possibly that genetic studies might no longer have to wait through months of winter and fall to rush through collection in the spring and summer. This may mean that research will not be limited to just the leaves that can be reached. The results of this study suggest that there is the possibility that the cambium can be used as a replacement for the live leaves.