Using acetone for rapid PCR‐amplifiable DNA extraction from recalcitrant woody plant taxa

Premise Quick and effective DNA extraction from plants for subsequent PCR amplification is sometimes challenging when working across diverse plant taxa that may contain a variety of inhibitory compounds. Time‐consuming methods may be needed to overcome these inhibitory effects as well as the effects of various preservation and collection methods to extract DNA from leaf samples. Our objective was to develop a rapid DNA extraction protocol that could be used with diverse plant taxa to produce high‐quality DNA suitable for downstream PCR applications. Methods and Results We tested the efficacy of acetone in extracting DNA from fresh, frozen, oven‐dried, acetone‐fixed, and herbarium leaf material of 22 species from 16 woody and herbaceous plant families. An improved simplified DNA extraction protocol was developed using acetone‐fixed leaf material. The addition of 1% sodium dodecyl sulfate solution resulted in the optimal extraction from all tissue samples. The DNA resulting from the extraction protocol was readily amplified using real‐time PCR assays. Conclusions The protocol described here resulted in the extraction of DNA from recalcitrant plant species that was of sufficient quality and quantity for PCR amplification, as indicated by the low threshold cycle values from real‐time assays. This method is simple, fast, and cost‐effective, and is a reliable tool for extracting high‐quality DNA from plant material containing PCR inhibitors.

favorable results from the simple acetone-based DNA extraction in S. albidum, plant samples from 12 diverse families (Aquifoliaceae, Buxaceae, Fabaceae, Fagaceae, Lauraceae, Oleaceae, Orchidaceae, Pinaceae, Poaceae, Rosaceae, Sapindaceae, and Solanaceae; Table  2) were subsequently tested using freshly collected leaves fixed in acetone. Eight additional plant species were tested using herbarium specimens (Appendix 1) from the U.S. National Arboretum ( Table  2). These taxa were selected based on their diversity, ensuring a representation of woody and herbaceous material, and the availability of material for sampling.

DNA extraction and quantification
A detailed protocol for DNA extraction and quantification is presented in Appendix 2. A clean cork borer sterilized with 95% ethanol was used to take three disks (5 mm in diameter, 5-10 mg each) of leaf material. The leaf disks were placed into a 1.5-mL microcentrifuge tube containing 0.5-1.0 mL of 100% acetone (enough to fully immerse the leaf tissue) at room temperature. The acetone was changed until the leaves turned white, after which the samples were stored at room temperature in the acetone until the DNA extraction. A single acetone-fixed leaf disk was air-dried, ground into small pieces using a metal rod in a 1.5-mL centrifuge tube, and incubated in buffer (200 µL of 1× TE with 1% SDS) at 90°C for 20 min. The tubes were vortexed twice during the incubation and then chilled on ice for 5 min. The tubes were centrifuged at 14,000 rpm for 5 min, after which the supernatant was transferred to a new 1.5-mL tube. An equal volume of 100% acetone was added and mixed by vortexing, and the mixture was then centrifuged at 6000 rpm for 1 min. The supernatant was discarded, and the resulting pellet was resuspended in 100 µL of nuclease-free water. Three samples from each DNA extraction were quantified using the Qubit dsDNA HS Assay kit and the Qubit 3.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), following the manufacturer's instructions. The average Qubit-based concentration was used to calculate the total amount of DNA extracted from the initial sample.
Real-time PCR assays were performed as three replicates per sample in a 10-µL final reaction volume using a Bio-Rad CFX96 Touch Real-Time System C1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, California, USA). The PCR mixture contained 0.5 µL of each 10-µM primer, 5.0 µL of Precision Melt Supermix (Bio-Rad Laboratories), 2.0 µL of nuclease-free water, and 1-2 ng of template DNA. The PCR profile consisted of 5 min of preheating at 95°C, followed by 40 cycles of 10 s of denaturation at 95°C, 10 s of annealing at 52°C, and 10 s of extension at 72°C. The plant universal primer pair ITS-p3/u4 was used to test the amplification of DNA from all plant samples, and ITS-3/4 was also used to test the amplification of DNA from herbarium specimens.

DNA quality and quantity
Experiments using S. albidum indicated that oven-dried samples resulted in the highest DNA yield (432 ng), which was 1.6 and 2.8 times greater than the yield from fresh and fixed leaf disk samples, Note: C t = threshold cycle; ITS-p3/u4 = internal transcribed spacer (ITS) of nuclear ribosomal DNA plant universal primer pair; SAFG-57 = Sassafras albidum transcriptomederived simple sequence repeat primer pair. a n = 3, mean ± SD. b Using 2 ng DNA per reaction. respectively (Table 1). Quantification with real-time PCR showed similar threshold cycle (C t ) values from all three sample preparation methods (Table 1). When tested on 13 additional plant taxa, the acetone and SDS extraction method from fresh tissue resulted in DNA yields of 11 ng to 1080 ng, with successful PCR amplification from all samples tested ( Table 2). The real-time PCR assays were performed with 1-2 ng of template DNA for each sample, and all species had C t values <35, indicating a reliable amplification efficiency. Extraction with acetone and SDS also resulted in amplifiable DNA from the recalcitrant woody plant species (S. albidum, Tsuga chinensis (Franch.) E. Pritz. in Diels, Picea orientalis (L.) Peterm., Prunus serrulata Lindl., Acer saccharum Marshall). The eight herbarium specimens yielded DNA using the acetone and SDS method, but the DNA was not consistently amplified using the primer pair ITS-p3/u4 (data not shown). Amplification using the primer pair ITS-3/4 was more consistent, but from only four of the eight samples (Pinus pinceana Gordon, Panicum virgatum L., Salix caroliniana Michx., and Solanum oplocense Hawkes) ( Table 2). The herbarium samples that yielded no amplification product likely had significantly degraded DNA.

CONCLUSIONS
We developed a simplified and quick DNA extraction method for herbaceous, woody, and herbarium plant specimens that uses only three chemical reagents (TE buffer, acetone, and SDS) and a single 5-mm-diameter leaf disk per sample without additional alcohol precipitation. The quality and quantity of the resulting DNA are sufficient for real-time PCR amplification and quantification of most samples, with the exception of some herbarium specimens.
Other recently reported methods have used similar modifications of existing protocols to optimize nucleic acid extraction from recalcitrant plants, but those methods rely on the modification of protocols developed for purchased extraction kits (Samarakoon et al., 2013) or use additional chemicals such as cetyltrimethylammonium bromide (CTAB), phenol, or chloroform for extraction, or salts and alcohol for multiple precipitation steps post-extraction (Samarakoon et al., 2013;Siegel et al., 2017;Barbier et al., 2019). The methods described here demonstrate the efficacy of tissue preservation and subsequent extraction in acetone with only the addition of 1% SDS, and without additional alcohol precipitation steps. This method provides a low-cost and rapid alternative to extract DNA from fresh, frozen, oven-dried, or herbarium tissue samples across many herbaceous and woody plant families, with yields and quality suitable for downstream PCR applications.

ACKNOWLEDGMENTS
The authors thank Ramon Jordan, Scott Warnke, John Hammond, and Harlan Svoboda (USDA, ARS, U.S. National Arboretum, Floral and Nursery Plants Research Unit) for providing plant samples used in this study.

AUTHOR CONTRIBUTIONS
F.E.G. analyzed data and wrote the manuscript. Y.H.G. designed and carried out experiments and analyzed data. M.R.P. analyzed data and wrote the manuscript. All authors gave their approval of the final manuscript before submission and publication.

DATA AVAILABILITY
The data provided in this study are fully discoverable, freely reusable, and citable. The raw sequence data used for the development of Sassafras albidum primers were obtained from the 1KP Transcriptomes Consortium available in the National Center for Biotechnology Information Sequence Read Archive (SRA) database under BioProject accession PRJEB4922.