Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.
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Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 251:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
CBOL Plant Working Group1 (2009) A DNA barcode for land plants. Proc Natl Acad Sci 106:12794–12797. https://doi.org/10.1073/pnas.0905845106
Fazekas AJ, Kuzmina ML, Newmaster SG, Hollingsworth PM (2012) DNA barcoding methods for land plants. In: Kress WJ, Erickson DL (eds) DNA barcodes. Humana Press, New York, pp 223–252
Freedman J, van Dorp L, Brace S (2018) Destructive sampling natural science collections: an overview for museum professionals and researchers. J Nat Sci Collect 5:21–34
Gilbert MTP, Moore W, Melchior L, Worebey M (2007) DNA extraction from dry museum beetles without conferring external morphological damage. PLoS One 2:1–4. https://doi.org/10.1371/journal.pone.0000272
Gugerli F, Parducci L, Petit RJ (2005) Ancient plant DNA: review and prospects. New Phytol 166:409–418. https://doi.org/10.1111/j.1469-8137.2005.01360.x
Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM (2004) Identification of birds through DNA barcodes. PLoS Biol 2:e312. https://doi.org/10.1371/journal.pbio.0020312
Hebert PDN, deWaard JR, Landry J-F (2010) DNA barcodes for 1/1000 of the animal kingdom. Biol Lett 6:359–362. https://doi.org/10.1098/rsbl.2009.0848
Keller GH, Manak MM (1993) DNA probes: background, applications, procedures. Stockton Press, New York
Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS One 2(6):e508. https://doi.org/10.1371/journal.pone.0000508
Kress WJ, Erickson DL, Jones FA, Swenson NG, Perez R, Sanjur O, Bermingham E (2009) Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama. Proc Natl Acad Sci 44:18621–18626. https://doi.org/10.1073/pnas.0909820106
Kuo LY, Li FW, Chiou WL, Wang CN (2011) First insights into fern matK phylogeny. Mol Phylogenet Evol 59(3):556–566. https://doi.org/10.1016/j.ympev.2011.03.010
Leonard JA (2008) Ancient DNA applications for wildlife conservation. Mol Ecol 17:4186–4196. https://doi.org/10.1111/j.1365-294X.2008.03891.x
Levin RA, Wagner WL, Hoch PC et al (2003) Family-level relationships of Onagraceae based on chloroplast rbcL and ndhF data. Am J Bot 90(1):107–115. https://doi.org/10.3732/ajb.90.1.107
Santos D, Ribeiro GC, Cabral AD, Sperança MA (2018) A non-destructive enzymatic method to extract DNA from arthropod specimens: implications for morphological and molecular studies. PLoS One 13:1–11. https://doi.org/10.1371/journal.pone.0192200
Satoh K, Shutoh K, Kurosawa T, Hayasaka E, Kaneko S (2018) Genetic analysis of Japanese and American specimens of Scripus hattorianus suggests its introduction from North America. J Plant Res 131:91–97. https://doi.org/10.1007/s10265-017-0976-7
Shepherd LD (2017) A non-destructive DNA sampling technique for herbarium specimens. PLoS One 12:e0183555. https://doi.org/10.1371/journal.pone.0183555
Wandeler P, Hoeck PEA, Keller LF (2007) Back to the future: museum specimens in population genetics. Trends Ecol Evol 22:634–642. https://doi.org/10.1016/j.tree.2007.08.017
Yukawa T (2016) Toxonomic notes on the Orchidaceae of Japan and adjacent regions. Bull Natl Mus Nat Sci, Ser B 42(3):103–111
This study was conducted under the project of Integrated Analysis of Natural History Collections for Conservation of Highly Endangered Species, the National Museum of Nature and Sciences, Tokyo, and supported by the 26th Fujiwara Natural History Foundation.
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Sugita, N., Ebihara, A., Hosoya, T. et al. Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves. J Plant Res 133, 133–141 (2020) doi:10.1007/s10265-019-01152-4
- DNA extraction
- Old specimen
- Soft leaf