Emerging methods in botanical DNA/RNA extraction

Analyses of nucleic acids (DNA and RNA) have become a staple tool for botanists to answer questions across wide variety disciplines, ranging from population genetics biogeography, ecology, development, microbiology, physiology, phylogenetics. The rise “next-generation” or “high-throughput” sequencing in particular has resulted reduced costs an explosion the number botanical studies using DNA RNA data (Egan et al., 2012). Yet, crucial step extracting these plant tissues can be extremely difficult is often overlooked under-emphasized. Although there are many options acid kits nearly countless papers (over 22,000 at time this special issue) referencing “modified” version Doyle (1987) cetyltrimethylammonium bromide (CTAB) extraction protocol, taxon-specific difficulties render methods ineffective. Troubleshooting remains major sink researchers' energy, potentially acting as barrier downstream analyses answering fundamental questions. Difficulties arise due factors such diversity volume secondary metabolites expressed by plants (Varma 2007), degradation during storage (Pyle Adams, 1989), contamination organisms microbiome (Trivedi 2022), need high-molecular-weight (Pollard 2018). Addressing issues requires knowledge both underlying chemistry involved each process requirements isolated product. 12 issue, “Emerging Methods Botanical DNA/RNA Extraction,” highlight current state extractions, including key challenges creative innovations that been developed circumvent address exciting most notable protocol CTAB-based approach (1987). However, researchers commonly refer “modified CTAB” approach, where various modifications highly varied, but without detailing what aspects were adjusted. To better understand alterations, first paper issue (Schenk 2023) reports results literature survey summary modified CTAB protocols. Schenk al. (2023) report provide recommendations eight steps protocol: tissue preparation, suspension, lysis, isolation, cleaning, elution, cleanup, quantification, with explanations why may require modification. Additionally, they four supplementary protocols appendices, which detail alterations lysis and/or steps. This review will allow troubleshoot their own extractions while also promoting repeatability transparency. Many designed “recalcitrant” plants, species whose chemical composition anatomy make much more difficult. Three articles specific ways improve prove recalcitrant different reasons. Recalcitrant contain high amounts hydrolytic enzymes exhibit nuclease activity, do silica-dried tissues. G. Johnson develop demonstrate utility ethanol, rather than silica gel, desiccant prior increase yield quality. Ethanol advantageous it act inhibit enzyme activity easier cell wall disruption. authors compare ethanol three examples: direct collection Vitaceae, pretreatment mangrove Rhizophora mangle (Rhizophoraceae), herbarium specimens 30 taxa representing nine families. increases quality quantity extracted DNA, especially appropriate proteinase digestion other treatments lysis. sclerophylly, Jones investigate effects mechanical disruption, time, leaflet age on concentration thick rigid leaves cycad genus Encephalartos. In cycads, leaf yielded greater concentrations when was manually disrupted mortar pestle vs. bead-based although purity unaffected disruption method. Tissue stored gel concentrations, no impact purity, compared freshly collected tissue. ranged less year decade, significant effect resulting concentration. Finally, reveal senescing young leaflets could adequately sufficient, pure applications. challenging, given presence polyphenols polysaccharides found woody species. Hadi Stacy test effectiveness isolation (along modifications) Metrosideros (Myrtaceae) differ anatomical makeup. They use absorbance ratios, integrity (RIN), success RNA-Seq only one popular kit effective taxa, further optimize high-quality suitable RNA-Seq. optimization extraction. As conduct research field settings, ability rapidly extract great advantage, not store well. Selz polymeric microneedle patches collect directly field. patches, originally drug delivery, produced lab taken After brief application surface, elution ultrapure water cleaning via spin column before ready patch tested standard nuclear chloroplast barcoding loci similar commercial its barcode represented National Center Biotechnology Information's GenBank database. offers flexible practical option remote locations. immediate useful collection, housed long-term provides opportunities historical contemporary existing specimens. Herbarium represent rich resource explore related climate change evolution, well providing access rare even extinct taxa. feasible, needs optimized account changes maximize small so used additional purposes future (Besnard 2018; Funk, Gouker previously published study procedure, species, specimen yield. While outperformed terms all enough subsequent experiments. Interestingly, entire set, (commercial kit, acetone, CTAB) trend within old (before 1960) new (after samples, species-specific differences between existed. Different unique affect acids. Two explicitly methods, Carey experimentally how additives incubation considerations genera. Based results, recommend shorter cooler periods fresh tissue) possible. Recently, moved toward storing frozen samples along typical samples. McAssey creatively set—the Hawaiian Plant Library, 1994–2019—with accessions sheets method A comparison paired revealed obtained significantly fragmented freezer, consequently lower-quality assemblies. Nevertheless, recovery genes short-read high-throughput affected method; instead, had substantial gene recovery. collections continue extraction, adding practice herbaria our evidence biodiversity future. Environmental (eDNA) assess taxonomic present environmental sources, air organismal products (like feces honey), water, soils (M. D. 2023), microbiome, i.e., biotic communities 2022). combined eDNA (eDNA metabarcoding, metagenomics) expanding potential identify (Deiner 2017). developments, technologies describe being applied diverse types. For example, Guillen-Otero ITS 18S rRNA metabarcoding characterize fungal exists ferns lycophytes. conducted relatively challenging tissue—root tissue—to arbuscular mycorrhizal fungi. measured lycophytes important insights into recent common ancestor shared angiosperms. Long-read platforms offer capability generating sequences tens hundreds thousands base pairs. These platforms, companies PacBio (Menlo Park, California, USA) Oxford Nanopore Technologies (Oxford, United Kingdom), sometimes referred “third-generation” technologies. Long reads helped facilitate transition assembly short (50–200 bp length) efficient means whole genome sequencing, 800 genomes sequenced (Marks 2021). Moreover, heavy transposable elements (TEs), long-read technology expand breadth (Shahid Slotkin, 2020). (fragments 50 kbp longer), generally modification obtaining fragment lengths little degradation. end, De La Cerda series straightforward (including beads cut intact pipette tips) nanopore enable evolutionary Calochortus, large genomes. Furthermore, sodium dodecyl sulfate–based promote less-contaminated critical Any length result run, primary concern working clades known having complementary fashion, Kang performed set spanning 18 orders. Testing continues choice studies. combines (to reduce organellar DNA) method, problems associated metabolites. novel longer fragments contamination. Importantly, recovered fraction long reads, variability among presumably varying levels compounds naturally leaves. fern lineages notoriously genomes, necessitates ensure sufficient quantities complete sequencing. Xie presented two yield: strategy prevent shearing nuclei substantially larger DNA. established preventing improves performance (Gong 2019), introduce that, requiring input tissue, generates order magnitude per approaches. (2023), read N50 scores >14 (Wickell 2021; Rahmatpour 2023). Overall, uses questions, cutting-edge generate data, consider all-important concerns, source materials, solutions molecules. We hope help readers protocols, toolkits, frontiers enabled data. N.M. prepared draft manuscript. All provided select article summaries reviewing editing assistance approved final thank submitting work reviewers lending expertise time. Thank you Applications Sciences editor-in-chief Dr. Briana L. Gross managing editor Beth Parada invaluable throughout editorial process.