A fast, flexible and inexpensive protocol for DNA and RNA extraction for forest trees
Abstract
Aim of the study: DNA and RNA extraction are still one of the most important and challenging steps of many molecular genetics applications such as Next-Generation Sequencing technologies. In this study, traditional laboratory preparation protocols and commercially available nucleic acids extraction kits’ features were combined into a procedure suitable for extraction of either DNA or RNA in 96-well plate format at high throughput.
Area of study: The study covers forest tree species from the United States of America.
Materials and methods: The DNA and RNA protocol were tested on 27 species, including especially recalcitrant forest tree species, from five angiosperm and three gymnosperm families. DNA was also extracted from stored (from 2 to 6 years) silica-dried samples of 11 species of Pinaceae.
Main results: The spectrophotometric analysis of DNA and RNA showed that gymnosperms yielded lower quantity, but higher quality nucleic acids than angiosperms which have variable results among species. The quantity and quality of DNA from stored samples were generally lower than fresh silica-dried samples. The RNA results showed high-enough yield (6.6 to 8.8 RIN) for downstream analyses.
Research highlights: It was demonstrated that high quality and high molecular weight nucleic acids for Next-Generation Sequencing applications can be isolated from hundreds of samples from a wide range of taxonomic groups. The new protocol has features similar to both traditional laboratory and commercial extraction kits; is easy to set up in any molecular research laboratory, can be applied to a large number of samples (hundreds) in a working day, uses inexpensive reagents and supplies, and is compatible with automation.
Key words: Angiosperms; gymnosperms; isolation protocol; nucleic acids.
Downloads
References
Abril N, Gion JM, Kerner R, Muller-Starck G, Cerrillo RMN, Plomion C, Renaut J, Valledor L, Jorrin-Novo JV, 2011. Proteomics research on forest trees, the most recalcitrant and orphan plant species. Phytochemistry 72: 1219-1242. https://doi.org/10.1016/j.phytochem.2011.01.005
Ashfaq M, Asif M, Anjum ZI, Zafar Y, 2013. Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae). Mol Ecol Resour 13: 573-582. https://doi.org/10.1111/1755-0998.12089
Barzegari A, Vahed SZ, Atashpaz S, Khani S, Omidi Y, 2010. Rapid and simple methodology for isolation of high-quality genomic DNA from coniferous tissues (Taxus baccata). Mol Biol Rep 37: 833-837. https://doi.org/10.1007/s11033-009-9634-z
Bashalkhanov S, Rajora OP, 2008. Protocol: A high-throughput DNA extraction system suitable for conifers. Plant Methods 4: 20. https://doi.org/10.1186/1746-4811-4-20
Buermans HPJ, den Dunnen JT, 2014. Next generation sequencing technology: Advances and applications. Biochim Biophys Acta 1842: 1932-1941. https://doi.org/10.1016/j.bbadis.2014.06.015
Canales J, Bautista R, Label P, Gomez-Maldonado J, Lesur I, Fernandez-Pozo N, Rueda-Lopez M, Guerrero-Fernandez D, Castro-Rodriguez V, Benzekri H et al., 2014. De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology. Plant Biotechnol J 12: 286-299. https://doi.org/10.1111/pbi.12136
CCDB protocols. Glass fiber plate DNA extraction protocol for plants, fungi, echinoderms and mollusks (www.ccdb.ca).
Chang S, Puryear J, Cairney J, 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113-116. https://doi.org/10.1007/BF02670468
Chase MW, Hills HH, 1991. Silica gel: An ideal material for field preservation of leaf samples for DNA studies. Taxon 40: 215-220. https://doi.org/10.2307/1222975
Chhatre VE, Byram TD, Neale DB, Wegrzyn JL, Krutovsky KV, 2013. Genetic structure and association mapping of adaptive and selective traits in the east Texas loblolly pine (Pinus taeda L.) breeding populations. Tree Genet Genomes 9: 1161-1178. https://doi.org/10.1007/s11295-013-0624-x
Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ, 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299. https://doi.org/10.1021/bi00591a005
Chomczynski P, Sacchi N, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159. https://doi.org/10.1016/0003-2697(87)90021-2
Claros MG, Canovas FM, 1998. Rapid high quality RNA preparation from pine seedlings. Plant Mol Biol Rep 16: 9-18. https://doi.org/10.1023/A:1007473906327
Csaikl UM, Bastian H, Brettschneider R, Gauch S, Meir A, Schauerte M, Scholz F, Sperisen C, Vornam B, Ziegenhagen B, 1998. Comparative analysis of different DNA extraction protocols: A fast, universal maxi-preparation of high-quality plant DNA for genetic evaluation and phylogenetic studies. Plant Mol Biol Rep 16: 69-86. https://doi.org/10.1023/A:1007428009556
Dellaporta SL, Wood J, Hicks, JB, 1983. A plant DNA mini preparation: Version II. Plant Mol Biol Rep 1: 19-21. https://doi.org/10.1007/BF02712670
Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19: 11-15.
Doyle JJ, Doyle JL, 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15. https://doi.org/10.2307/2419362
Drabkova L, Kirschner J, Vlcek C, 2002. Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of Juncaceae. Plant Mol Biol Rep 20: 161-175. https://doi.org/10.1007/BF02799431
Healey A, Furtado A, Cooper T, Henry RJ, 2014. Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 10: 21. https://doi.org/10.1186/1746-4811-10-21
Ivanova NV, deWaard JR, Hebert PDN, 2006. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol Ecol Notes 6: 998-1002. https://doi.org/10.1111/j.1471-8286.2006.01428.x
Ivanova NV, Fazekas AJ, Hebert PDN, 2008. Semi-automated, membrane-based protocol for DNA isolation from plants. Plant Mol Biol Rep 26: 186-198. https://doi.org/10.1007/s11105-008-0029-4
Kesanakurti PR, Fazekas AJ, Burgess KS, Percy DM, Newmaster SG et al., 2011. Spatial patterns of plant diversity below-ground as revealed by DNA barcoding. Mol Ecol 20: 1289-1302. https://doi.org/10.1111/j.1365-294X.2010.04989.x
Khan MA, Korban SS, 2012. Association mapping in forest trees and fruit crops. J Exp Bot 63: 4045-4060. https://doi.org/10.1093/jxb/ers105
Kiefer E, Heller W, Ernst D, 2000. A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Mol Biol Rep 18: 33-39. https://doi.org/10.1007/BF02825291
Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI, 1997. A simple and rapid method for isolation of high-quality genomic DNA from fruit trees and conifers using PVP. Nucleic Acids Res 25: 1085-1086. https://doi.org/10.1093/nar/25.5.1085
Lefort F, Douglas GC, 1999. An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus. Ann For Sci 56: 259-263. https://doi.org/10.1051/forest:19990308
Le Provost G, Herrera R, Ap Paiva J, Chaumeil P, Salin F, Plomion C, 2007. A micromethod for high throughput RNA extraction in forest trees. Biol Res 40: 291-297. https://doi.org/10.4067/S0716-97602007000400003
Lutz KA, Wang W, Zdepski A, Michael TP, 2011. Isolation and analysis of high-quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing. BMC Biotechnol 11: 54. https://doi.org/10.1186/1472-6750-11-54
Neale DB, Kremer A, 2011. Forest tree genomics: growing resources and applications. Nat Rev Genet 12: 111-122. https://doi.org/10.1038/nrg2931
Ostrowska E, Muralitharan M, Chandler S, Volker P, Hetherington S, Dunshea F, 1998. Optimizing conditions for DNA isolation from Pinus radiata. In Vitro Cell Dev Biol Plant 34: 108-111. https://doi.org/10.1007/BF02822773
Plomion C, Chancerel E, Endelman J, Lamy JB, Mandrou E, Lesur I, Ehrenmann F, Isik F, Bink MC, Heerwaarden J et al., 2014. Genome-wide distribution of genetic diversity and linkage disequilibrium in a mass-selected population of maritime pine. BMC Genomics 15: 171. https://doi.org/10.1186/1471-2164-15-171
Reynolds MM, Williams CG, 2004. Extracting DNA from submerged pine wood. Genome 47: 994-997. https://doi.org/10.1139/g04-045
Sarkinen T, Staats M, Richardson JE, Cowan RS, Bakker FT, 2012. How to open the treasure chest? Optimising DNA extraction from herbarium specimens. PLoS ONE 7: e43808. https://doi.org/10.1371/journal.pone.0043808
Semagn K, 2014. Leaf tissue sampling and DNA extraction protocols. In: Molecular Plant Taxonomy: Methods and Protocols. Methods in Molecular Biology; Besse P (ed). vol. 1115, Humana Press, Springer Science + Business Media, New York. https://doi.org/10.1007/978-1-62703-767-9_3
Shepherd M, Cross M, Stokoe RL, Scott LJ, Jones ME, 2002. High-throughput DNA extraction from forest trees. Plant Mol Biol Rep 20: 425a-425j. https://doi.org/10.1007/BF02772134
Staats M, Erkens RHJ, van de Vossenberg B, Wieringa JJ, Kraaijeveld K, Stielow B, Geml J, Richardson JE, Bakker FT, 2013. Genomic treasure troves: Complete genome sequencing of herbarium and insect museum specimens. PLoS ONE 8(7): e69189. https://doi.org/10.1371/journal.pone.0069189
Telfer E, Graham N, Stanbra L, Manley T, Wilcox P, 2013. Extraction of high purity genomic DNA from pine for use in a high-throughput Genotyping Platform. New Zeal J For Sci 43: 3. https://doi.org/10.1186/1179-5395-43-3
Tibbits JFG, McManus LJ, Spokevicius AV, Bossinger G, 2006. A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees. Plant Mol Biol Rep 24: 81-91. https://doi.org/10.1007/BF02914048
Valledor L, Escandon M, Meijon M, Nukarinen E, Canal MJ, Weckwerth W, 2014. A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms. Plant J 79: 173-180. https://doi.org/10.1111/tpj.12546
Van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C, 2014. Ten years of next-generation sequencing technology. Trends Genet 30: 418-426. https://doi.org/10.1016/j.tig.2014.07.001
Wang XR, Szmidt AE, 2001. Molecular markers in population genetics of forest trees. Scand J Forest Res 16: 199-220. https://doi.org/10.1080/02827580118146
Whitlock R, Hipperson H, Mannarelli M, Burke T, 2008. A high-throughput protocol for extracting high-purity genomic DNA from plants and animals. Mol Ecol Res 8: 736-741. https://doi.org/10.1111/j.1755-0998.2007.02074.x
Xin Z, Chen J, 2012. A high throughput DNA extraction method with high yield and quality. Plant Methods 8: 26. https://doi.org/10.1186/1746-4811-8-26
© CSIC. Manuscripts published in both the printed and online versions of this Journal are the property of Consejo Superior de Investigaciones Científicas, and quoting this source is a requirement for any partial or full reproduction.
All contents of this electronic edition, except where otherwise noted, are distributed under a “Creative Commons Attribution 4.0 International” (CC BY 4.0) License. You may read here the basic information and the legal text of the license. The indication of the CC BY 4.0 License must be expressly stated in this way when necessary.
Self-archiving in repositories, personal webpages or similar, of any version other than the published by the Editor, is not allowed.
