Successful extraction of DNA from plant tissues generally has been most successful from freshly field-collected tissue placed into either liquid nitrogen or, more commonly, into silica gel desiccant. When possible, the samples are then stored frozen in ultracold liquid nitrogen tanks or in boxes containing additional silica gel at −20°C to −80°C. Subsequent nucleic acid extractions most commonly have involved either some form of a cetyltrimethylammonium bromide (CTAB)–based technique (Doyle and Doyle, 1987) or use of a QIAGEN DNeasy kit (QIAGEN, Venlo, The Netherlands). However, previous research has also shown success with an alternative genomic DNA extraction method using Whatman FTA PlantSaver Cards (Whatman, Maidstone, United Kingdom), with effectiveness for agricultural plant species, entomology, mycology, and other food sciences (Suzuki et al., 2006; Marques et al., 2010; Adugna et al., 2011; Bujang et al., 2011; Chandrashekara et al., 2012). Nevertheless, remarkably little research has been done to test the effectiveness of this extraction method on non-agricultural plant species. Additionally, it has been shown that methods employing DNeasy kits and/or CTAB-based methods may not be optimal for all plant genera and families because inhibitors can be coprecipitated with the DNA (Bustin and Nolan, 2004; Adugna et al., 2011). Therefore, it is proposed that alternative extraction methods, such as use of the FTA cards, be tested as to whether they are more effective for some plant genera.
To help gain a better understanding of the potential uses and limitations of FTA card extraction, this study assessed FTA card–extracted DNA quality in terms of concentration, spectral absorption, degree of fragmentation, and amplification and sequencing ability on a wide phylogenetic range of non-agricultural species. The data collected using these methods helped to indicate which plant species may be most compatible with the FTA card extraction method. Both successful and failed extractions provided valuable insights into the potential advantages and limitations of this alternative extraction method.
MATERIALS AND METHODS
Collection —Samples from 15 phylogenetically diverse taxa possessing varying leaf characteristics were collected from the following vascular plant families: Apocynaceae, Aquifoliaceae, Asteraceae, Cactaceae, Cyperaceae, Fabaceae, Lamiaceae, Magnoliaceae, Oleaceae, Oxalidaceae, Poaceae, Typhaceae, Vitaceae, Pinaceae, and Aspleniaceae (Table 1). Collection of samples was completed on the morning of 23 June 2015, alongside a gravel utility road, sloping hillsides, and free-standing trees in Suitland, Maryland, USA. Voucher specimens for each species were deposited in the National Museum of Natural History's Herbarium (US), the University of Illinois at Urbana-Champaign Plant Biology Herbarium (ILL), and the Chicago Botanic Garden (CHIC).
Samples were preserved using Whatman FTA PlantSaver Cards and in silica gel (Flower Drying Type A silica with indicator; AGM Container Controls, Tucson, Arizona, USA). Samples were applied to the FTA cards directly in the field, then stored at room temperature. To make the plant print, a ceramic pestle was used like a hammer to smash the leaf tissue onto the card paper (Fig. 1). Enough plant prints were made for seven replicate extractions to be performed for each species.
Additionally, 2–3 in2 of leaf tissue were collected into individual silica gel– containing bags to be extracted later, using the CTAB-based technique on the AutoGen DNA isolation system (AutoGen, Holliston, Massachusetts, USA). Bags were stored in a plastic box containing additional silica gel at room temperature prior to extraction and then moved to a −20°C freezer for long-term storage.
FTA card extraction—After collecting the samples on the FTA cards, small punched disks were removed from the sample cards followed by a series of washes on the disks. The protocol used was closely modeled after the method outlined in Adugna et al. (2011), with a modified disk size and centrifugation added after the incubation period to reduce bubbles. Eight disks, 2.0 mm in diameter, were used in this study so as to have a comparable total disk surface area to that found in Adugna et al. (2011). Additionally, the protocol of Adugna et al. called for centrifugation only after the addition of TE in the last step of the protocol. In our study, plates were centrifuged at 6200 rpm for 2 min at 4°C in between each purification wash.
Using the 2.0-mm-diameter Harris Micro Punch and Mat (Whatman), 56 disks were removed from each specimen's plant-pressed FTA card. A 96-well, Square V-Bottom 2-mL Assay Block (Costar, Corning Inc., Corning, New York, USA) was used with eight punched disks placed in each well. The disks were only added to the innermost wells (rows 3–10 out of 12) of the plates for more effective and cleaner transfer from one plate to another. Disk punches were taken from the leaves' darker print areas on the cards, which were presumed to contain the most concentrated residue.
Once disks had been collected from each plant, a series of washes were employed according to the manufacturer's protocol. First, 400 µL of FTA purification reagent was added to each well. The plate of samples was then covered with Microseal ‘F’ foil seals (Bio-Rad, Hercules, California, USA) to reduce contamination, vortexed, and incubated at room temperature for 4 min before centrifugation at 6200 rpm for 2 min at 4°C. The supernatant was removed and the plates were then centrifuged a second time before the foil was removed. The FTA purification reagent wash was repeated once, followed by two low TE buffer washes. After completing the four washes, the remaining supernatant was removed. The samples were left to dry at room temperature for 20 min. The disks were then transferred to a new plate and centrifuged to separate them from as much remaining supernatant as possible. Any punches not in the new plate were manually transferred using cleaned forceps. Finally, 80 µL of TE was added to the plate containing the washed disks. The plate was centrifuged at 6200 rpm for 1 min and then incubated for 5 min at 95°C. The paper disks were left in the sample wells with the TE and eluted DNA.
Species sampled in this study. All specimens were collected in Camp Springs, Maryland, USA (GPS coordinates 38°50′40.4″N, 76°56′17.4″W). Triplicate vouchers were made for deposit at the National Museum of Natural History′s Herbarium (US), the University of Illinois at Urbana-Champaign Plant Biology Herbarium (ILL), and the Chicago Botanic Garden (CHIC). Samples are organized alphabetically by family name.
CTAB-based extraction—For each replicate, a 1.0-cm2-sized piece of dried and shredded leaf tissue was added to a 2.0-mL tube containing 2.3-mm-diameter zirconia-silica beads and 1.0-mm-diameter glass beads (BioSpec, Bartlesville, Oklahoma, USA). The tissue was then macerated using the TissueLyser (QIAGEN) at 30 Hz for 30 s before 400 µL of CTAB, warmed to 65°C, was added. The Cactaceae samples produced a viscous gel, so 75 µL of 20 mg/mL Proteinase K and 500 µL of CTAB were added to further clean the sample. All samples were incubated overnight in a rotary incubator at 65°C and 150 rpm.
The resulting lysate solutions were centrifuged at 13,000 rpm for 5 min and 300 µL of supernatant was transferred to each well of a 96-well AutoGen plate. DNA extraction was completed using the automated DNA isolation system AutoGenprep 965 (AutoGen) as outlined by the manufacturer's protocol, and the final pellets were resuspended in 80 µL of TE buffer.
Quantification and quality assessment —To assess the quality of the extractions generated with the two techniques, 260/280 nm absorbance ratios and fluorometric determinations of DNA concentration were performed using a Synergy HT Microplate Reader (BioTek, Winooski, Vermont, USA). The Quant-iT Broad-Range dsDNA Assay Kit (Invitrogen, Waltham, Massachusetts, USA) was used with the Synergy Microplate Reader according to the manufacturer's protocol (Fig. 2). The DNA size range and degree of fragmentation were determined using gel electrophoresis (Fig. 3). The samples (10 µL) were run alongside a HiLo DNA size standard (Minnesota Molecular, Minneapolis, Minnesota, USA) on a 1.5% SeaKem Agarose LE gel in 1× SIB (Brody and Kern, 2004). The loading dye used to prepare these electrophoretic separations contained a 1 : 1000 dilution of Gel Red fluorescent nucleic acid stain (Biotium, Fremont, California, USA).
PCR amplification and DNA sequencing —To compare the amplification success of DNA extracted with the two methods, a portion of the low-copy nuclear gene At103, the nuclear ribosomal intergenic spacer (ITS), and the plastid ribulose-bisphosphate-dismutase large subunit (rbcL) were amplified. Each amplification reaction contained 2.5 µL of FTA card–extracted sample or 2.5 µL of 1 : 50 diluted CTAB-extracted sample. The At103 region was amplified with forward primer CTTCAAGCCMAAGTTCATCTTCTA and reverse primer TTGGCAATCCATTGAGGTACATNGTM (Li et al., 2008), and the ITS re