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Investigating Leaf Preservation Methods to Optimize DNA Concentration for Genomic Analysis of Populus Species.
McFadden, Shannon
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Denver, CO
Metropolitan State University of Denver
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Collected for Auraria Institutional Repository by the Self-Submittal tool. Submitted by Matthew Mariner.
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Faculty mentor: Bissell, Erin
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Major: Biology

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Auraria Institutional Repository
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Auraria Library
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50 70% of angiosperm species have arisen from hybridization making hybrid genomic studies a valuable insight into this evolutionary process 1 . In order to perform these studies, high molecular grade DNA must be obtained. The method of storing leaf samples can affect the quality of the DNA yield. Chemical preservation (FAA, ethanol, etc.) degrades DNA 2 . Dry storage (with silica) and freezing methods have been successful but their efficacy is dependent on the characteristics of the species in question 2 . This study investigates the efficacy of two leaf storage methods, dry storage and freezing, on Populus deltoides , P. angustifolia, and their hybrid P. x acuminata , over the course of two years. Following extraction, DNA yield and quality will be evaluated based on preliminary spectrophotometry, PCR, and gel electrophoresis. Once identified, the best method can be used in large scale genomic studies of Populus , allowing them to be conducted in a timely manner but without having to process large quantities of fresh samples in a single session. INTRODUCTION Collection Location: Denver Botanic Gardens Chatfield Farms (Littleton, CO) September 2019 and October 2018 Three sites: C3, C4, and C5.2. Each plot contained all species in question. At all 3 sites 8 leaves were collected from a tree of each species. Storage 4 samples were taken per leaf using a standard hole punch (see Figure 1). Dry storage: Samples were placed in mesh bags with silica beads (10:1 ratio of beads to sample), labeled, and stored at room temperature. Frozen storage: Samples were places in plastic bags, labels, and stored in the freezer at 18 degrees C. Extraction DNA extraction was conducted using QIAGEN DNeasy Plant Mini Kits. A cordless handheld homogenizer was used for disruption. 2018 samples extracted ~18mo after collection 2019 samples extracted ~6mo after collection. Quality Assessment Preliminary spectrophotometry U NanoDrop 2000 Polymerase chain reaction Using p rimer pairs: ITS u1/ITS u4 and ITS p5/ITS u4 Gel electrophoresis MATERIALS & METHODS The data shown in Figure 2 are preliminary spectrophotometry results of the DNA concentration, 260/280, and 260/230 ratios of all the P. angustifolia samples from 2019 and a small number of the samples from 2018. The average concentration of dried samples is 6.73 ( 1.91) ng/mL Average concentration of frozen samples is 5.98 ( 1.02) ng/mL Mean 260/280 ratio: dried 1.64 ( 0.20), frozen 1.49 ( 0.12) Mean 260/230 ratio: dried 1.53 ( 0.61), frozen 1.09 ( 0.47) We would like thank Metropolitan State University of Denver for awarding the Undergraduate Research Mini Grant as well as the Chemistry department for providing liquid nitrogen. We appreciate the cooperation of Denver Botanic Gardens at Chatfield Farms for granting access to their property to collect cottonwood leaf samples. Thanks also go to Dr. Petcoff and Dr. Ferrell for use of the cold storage dewar in their lab and Tracy Fielder for help with early experiments in tissue disruption. A special thank you also to Taylor Boyd for her assistance in tissue disruption. REFERENCES Biology Department at Metropolitan State University of Denver Shannon MacFadden , Faculty Advisor: Erin Bissell, PhD Investigating Leaf Preservation Methods to Optimize DNA Concentration for Genomic Analysis of Populus Species RESULTS 1. Martinsen, G. D., Whitham, T. G., Turek , R. J. & Keim , P. Hybrid Populations Selectively Filter Gene Introgression between Species Author ( s ): Gregory D . Martinsen , Thomas G . Whitham , Richard J . Turek and Paul Keim Published by : Society for the Study of Evolution Stable URL : 55 , 1325 1335 (2001). 2. Pyle, M. M. & Adams, R. P. IN SITU PRESERVATION OF DNA IN PLANT SPECIMENS . Taxon 38 , 576 581 (1989). 3. Chase, M. W. & Hills, H. H. Silica gel: An ideal material for field preservation of leaf samples for DNA studies. Taxon 40 , 215 220 (1991). 4. Cheng, T. et al. Barcoding the kingdom Plantae: new PCR primers for ITS regions of plants with improved universality and specificity. Mol. Ecol. Resour . 16 , 138 149 (2016). 5. Mulcahy, D. G. et al. Greater than X kb: A quantitative assessment of preservation conditions on genomic DNA quality, and a proposed standard for genome quality DNA. PeerJ 2016 , (2016). DISCUSSION The current data are strictly preliminary and only fully reflect nanodrop results from P. angustifolia samples stored for 6 months as well as a partial data set of the same species stored over 18 months. Extraction of the remaining samples too early to extrapolate any definitive conclusions from the current data, there are possible trends in the data that will need to be examined closer as more data is collected. Concentrations of DNA are overall similar using both methods indicating that storage method may not affect quantity. Modestly lower average of 260/280 and, more specifically, 260/230 ratios in frozen samples may suggest storage method affects quality. We need to collect more data for all species and run statistical analyses to assess whether the differences are significant. 260/230 value < 1.6 is indicative of polysaccharide contaminations such as carbohydrate carryover (common in plants). 260/280 value < 1.6 is indicative of protein contamination. Freezing samples is generally problematic in species high in phenols, flavonoids, and other readily oxidized compounds due to biochemical responses 3 . If this trend continues, it might be because Populus species contain these compounds, making dry storage the superior method for sample preservation. FUTURE DIRECTIONS Once DNA is extracted from the remaining samples, PCR and gel electrophoresis will expand our understanding on whether either method produces a higher quality product. Two sets of primers have been identified to assess DNA quality moving forward: ITS u1/ITS u4 and ITS p5/ITS u4. Amplify the ITS region of nuclear DNA. Most commonly used in plant barcoding as a valuable tool for plant systematic studies at the species level 4 . Primers designed to provide universal amplification high universality across plant species while minimizing amplification of fungi 4 . Gel electrophoresis on agar is the most widely used method to quantify DNA 5 and will provide higher precision in quality assessment. Figure 1: Leaves collected in October 2018, shown after hole punch sampling. P. deltoides (Plains cottonwood) Populus x acuminata ( Lanceleaf cottonwood) Populus angustifolia (Narrowleaf cottonwood) Figure 2: Preliminary results comparing DNA concentration (left), 260/280 ratios (middle), and 260/230 ratios (right) for P. angustifolia samples collected in October 2018 and September 2019. ACKNOWLEDGEMENTS Shannon MacFadden collecting leaf samples from Populus angustifolia at Denver Botanic Gardens Chatfield Farms in September 2019 (Photo credit: Erin Bissell)