Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.

PREMISE OF THE STUDY: Here we present a series of protocols for RNA extraction across a diverse array of plants; we focus on woody, aromatic, aquatic, and other chemically complex taxa. METHODS AND
RESULTS: Ninety-one taxa were subjected to RNA extraction with three methods presented here: (1) TRIzol/TURBO DNA-free kits using the manufacturer's protocol with the addition of sarkosyl; (2) a combination method using cetyltrimethylammonium bromide (CTAB) and TRIzol/sarkosyl/TURBO DNA-free; and (3) a combination of CTAB and QIAGEN RNeasy Plant Mini Kit. Bench-ready protocols are given.
CONCLUSIONS: After an iterative process of working with chemically complex taxa, we conclude that the use of TRIzol supplemented with sarkosyl and the TURBO DNA-free kit is an effective, efficient, and robust method for obtaining RNA from 100 mg of leaf tissue of land plant species (Embryophyta) examined. Our protocols can be used to provide RNA of suitable stability, quantity, and quality for transcriptome sequencing.

Next-generation sequencing (NGS) offers numerous research opportunities to the plant systematics and evolutionary biology communities (e.g., Godden et al., 2012; Strickler et al., 2012; Soltis et al., 2013). To apply NGS to whole transcriptome sequencing, high-quality RNA must be reliably obtained, often from diverse taxa. However, high levels of compounds such as flavonoids, tannins, waxes, and other secondary metabolites found in the tissues of aromatic, woody, and aquatic plants can make it difficult to extract RNA in sufficient quantity and quality for NGS. To circumvent these problems, many different methods have been developed for RNA extraction from plant tissues (e.g., Johnson et al., 2012; Yockteng et al., 2013; Zhu et al., 2013).

During our involvement in the 1KP project (One Thousand Plants Initiative, see www.onekp.com), we developed two RNA isolation protocols that were used to obtain high-quality and -quantity RNA from a diverse set of plant species. Here we present two protocols (Options 1 and 2) in detail and compare their success rate with a third protocol (Option 3) that is standardly used for many species (e.g., Buggs et al., 2009; Johnson et al., 2012). An earlier version of our Option 1 can be found in the appendix of Johnson et al. (2012) in Protocol 14, but is presented here with further refinements that improve RNA quality and quantity. Option 2 is presented for the first time.

In this manuscript, we quantify RNA extraction success among the methods with Bioanalyzer metrics, not transcriptome sequencing success as was done in Johnson et al. (2012), by quantifying number of scaffolds and resulting assemblies. An extraction was considered successful if it met the quantity and quality set by the 1KP consortium agreement: ≥30 μg of total RNA, RNA integrity number (RIN) higher than 5, and rRNA ratio (26S/18S) greater than 1. Although Johnson et al. (2012) conclude that transcriptome assemblies were mostly dependent on the NGS sequencing platform, RNA quality (RIN and optical density [OD] ratios) and quantity also had significant effects. The two preferred protocols presented here result in RNA isolations of the quality and quantity required for transcriptome sequencing in a wide range of plant taxa.

METHODS AND RESULTS

Selection of material

The plants selected for the 1KP project were meant to circumscribe the entire green plant clade (Viridiplantae), including chlorophyte algae (see Johnson et al., 2012; Matasci et al., 2014). RNA from the majority of taxa in the 1KP project were extracted with protocols outlined in Johnson et al. (2012). The protocols presented here were developed to handle samples for which those methods had failed to provide good-quality RNA extractions. Most are angiosperms and many have characteristics (aromatic, woody, aquatic) known to present challenges to RNA extraction (Appendix 1).

Overview of protocols

Option 1 is the manufacturer’s protocol for RNA extraction with TRIzol (Ambion, Life Technologies, Carlsbad, California, USA), with the addition of sarkosyl to the extraction solution. Option 2 combines traditional cetyltrimethylammonium bromide (CTAB) extraction (Doyle and Doyle, 1987) followed by the use of TRIzol with sarkosyl (i.e., CTAB followed by Option 1). Both Option 1 and 2 used the TURBO DNA-free kit (Ambion) for DNA digestion, which proved to be superior for RNA stability compared to the on-column digestion (Appendix 2 for taxa that degraded [gray cells]). Option 3 combines the CTAB extraction method followed by the use of half of the QIAGEN RNeasy Plant Mini Kit for on-column DNA digestion (QIAGEN, Valencia, California, USA). Option 3 was the protocol used during the initial phase of the 1KP project, and in other projects in our laboratory (e.g., Buggs et al., 2009), but after multiple failures, Options 1 and 2 were developed to deal with difficult plant materials. These methods are briefly outlined below, and detailed bench-ready protocols can be found in Appendices 3–6). A flow chart can be found in Fig. 1. Option 1 is recommended as a starting point when working with unstudied taxa because it has fewer steps. If this fails, researchers should then move to Option 2. The protocols here can be adapted to processing few samples or relatively high-throughput extractions of 12 to 24 samples at a time. The use of 2-mL microcentrifuge tubes and an automatic shaker for tissue pulverization is ideal (see Appendix 3 for details on tissue collection and processing). These methods can still be used with tissue pulverization in a mortar and pestle if an automatic shaker is not available or the tissue will not pulverize by beads alone, then transferred to the 2-mL tubes (i.e., some Poaceae and succulent species).

Fig. 1.

Flow chart describing the iterative process to successfully obtain high-quality and -quantity RNA using Option 1 and Option 2. (Option 3 is excluded, as it is not recommended.)

The addition of sarkosyl in the TRIzol extraction step in either Option 1 or 2 was integral for improving quality and yield in some taxa, but was not required for all taxa (data not shown). The addition of sarkosyl to lysis solutions is not new; it has been used in both plant and animal nucleic acid and protein isolations since the 1970s (Kingston, 2010) and is still used today for various applications (Huang et al., 2012). It is commonly recommended as an addition to isolation buffers containing guanidine (Kingston, 2010), an ingredient in the TRIzol solution. The addition of sarkosyl to the Ambion TRIzol extraction kit was used to deal with plants with high amounts of organic compounds or other complexities (typically indicated by a brown color in the aqueous layer). After centrifugation, the lightweight sarkosyl layer rested at the interface between the upper aqueous solution and the lower organic layer. Following TRIzol and sarkosyl extraction, a 100% chloroform extraction was always done once, sometimes twice, depending on whether a whitish interface was present between the organic and the aqueous layers.

RNA precipitation and pelleting

In all of the options, the method of RNA precipitation was similar (in Option 3, 5 M NaCl was also added). The tube containing the aqueous phase was filled with 100% isopropanol at room temperature, gently inverted, then incubated at −20°C for 10 min for Options 1 and 2, and 20 min for Option 3. Overnight incubation did not increase the yield, and the best RNA was obtained when no cloudiness or white precipitate was formed after addition of isopropanol. After incubation, in Options 1 and 2 the samples were centrifuged at 4°C for 20 min to pellet the RNA, typically producing pellets of 0.25–1 cm long (the expected size as indicated by Ambion). The RNA was not pelleted in Option 3. The pellet was washed with 75% ethanol and air-dried for no longer than 10 min (otherwise resuspension was difficult), and resuspended in RNase-free water by incubation at 37°C on an orbital shaker for 10 min. If the pellet was not dissolved by the end of this incubation period, in most cases, it was not worth proceeding, and the process was started from the beginning with a new sample (Fig. 1). For Option 3, if the NanoDrop (Thermo Scientific, Waltham, Massachusetts, USA) gave poor results (data not shown), the sample was also discarded. Therefore, these failed attempts do not have Bioanalyzer metrics as others listed in Appendix 2.

Extraction success: Quality, quantity, and stability

Extraction success was measured from Bioanalyzer metrics for the quality and quantity of RNA isolated (Agilent Technologies, Santa Clara, California, USA) (Fig. 2, Appendix 2, and Appendix S1 for those that were analyzed with the Bioanalyzer). Over the course of this study, 382 separate RNA extractions were attempted from leaf tissues, of which 138 were successful (Table 1). Many of the failures were due to the iterative process of developing the protocols presented here and multiple unsuccessful attempts with Option 3 before alternatives were sought. Table 1 summarizes the levels of extraction success of each method during this process, and the number of taxa for which RNA was successfully extracted in the end. Using our methods, we finally successfully extracted RNA from 77 unique taxa of 91 attempted (85%). Option 1 was tested on 74 of the 91 taxa in this data set, Option 2 was tested on 41 of the 91 taxa, Option 3 was used on 68 out of 91 taxa. Not all methods were tried on all taxa, as the goal of our work was to obtain pure RNA for the 1KP project as quickly as possible, not to test all methods across all taxa. Options 1 and 2 often succeeded with material that failed to yield RNA with Option 3 (Appendix 2). Nine taxa were unsuccessful in extraction with any of the three methods, perhaps due to the chemical composition of the plant, or perhaps tissue quality (Appendix 2). Two taxa yielded good-quality and -quantity RNA, but the library construction failed at BGI (Shenzen, China) (for details see Johnson et al. [2012] and Appendix 2). Most of the extractions that were partially successful with Option 3 were of low RNA quality, and often degraded (gray cells in Appendix 2), but the nondegraded isolations from Option 3 were sometimes ultimately suitable for transcriptome sequencing (Appendix 2).

Fig. 2.

Examples of Agilent 2100 Bioanalyzer spectra of total RNA showing improvement with Options 1 and 2 compared to Option 3. Each graph shows the intensity of the peaks of the ribosomal RNA subunits: nuclear large-28S, small-18S, cytoplasmic, mitochondrial, and chloroplastic (smaller subunits). The electrophoretic gel for each sample is shown to the right indicating the subunit bands or degradation (i.e., smear). nt = number of estimated nucleotides based on ladder; FU = fluorescence unit (i.e., intensity of peak). (A) Degraded ribosomal RNA subunits of Canella winterana (L.) Gaertn. extracted with Option 3 that resulted in an estimation of 27 μg of RNA, but the subunits are degraded. (B) A second example using Option 3, Muntingia calabura L., also shows an inflated quantity reading with degraded subunits. (C) Canella winterana, extracted with Option 1, indicating peaks for intact ribosomal RNA subunits. (D) Muntingia calabura extracted with Option 2.

Table 1.

Success of RNA extraction for each method. Success is defined by the Bioanalyzer results and final quantity of pure RNA that was sequenced. Final concentration was estimated from the Bioanalyzer and the known volume of the final extraction. Some samples were extracted multiple times and pooled (i.e., “repeats”). Note that most samples were initially tried with Option 3; those that failed were then attempted with Options 1 and 2. Thus, not all samples were extracted with all options.

Method	No. of successful RNA extractions/total RNA extractions tried	% of successful RNA extractions	No. of taxa with successful RNA extractions/tried taxa	% of taxa with successful RNA extractions	Avg. μg of total RNA
Option 1: TRIzol/TURBO DNA-free	56/119	47	34/74	46	51.1
Option 2: CTAB/ TRIzol/TURBO DNA-free	43/63	68	31/41	76	77.6
Option 3: CTAB/QIAGEN Kit/on-column digestion	39/200	20	18/68	26	33.8
Overall totals	138/382	36	83/183 (without repeats 77/91)	45 (without repeats 85)	Total average 54.2

Bioanalyzer metrics for the total quantity of RNA isolated, RIN, and OD ratio were not the same across the different extraction options (Fig. 3). Given that the data set was compiled as trial and error, but not formally designed for statistical measures, only the distribution of the data is shown (Appendix S1). For the purposes of the 1KP project, 30 μg of total RNA was requested for each taxon from BGI for a full transcriptome sequence (Johnson et al., 2012). However, today, 30 μg of RNA is not necessarily needed to generate transcriptomes. For the 18 taxa successfully extracted with Option 3 in this study, multiple extractions (2–4) had to be done and pooled to reach the desired amount (using ∼100 mg of frozen tissue per extraction). Out of the successful extractions made with Options 1 and 2, only 33% of the taxa gave less than 30 μg in the first extraction attempt, and at most two extractions were needed to obtain the quantity desired. The majority of the time for Option 2 (67% of the extractions), the quantity averaged 77 μg of RNA from only 100 mg of tissue in the first extraction attempt, so no pooling was needed. The maximum amount was 250 μg of RNA in one extraction.

Fig. 3.

Distribution of RNA quantity and quality. Box plots illustrate (A) total micrograms of RNA isolated by each method, (B) RNA integrity number resulting from each method, and (C) rRNA (OD) ratio resulting from each method. The error bars and the boxes indicate the quantiles of the data (JMP Pro 11.0, SAS Institute).

To NanoDrop or not

NanoDrop or similar equipment for RNA (or DNA) concentration measures can be used to obtain an estimate for total amount of nucleic acids. The NanoDrop cannot detect the presence of intact ribosomal-RNA subunits, and therefore is not able to detect if the RNA is degraded. Additionally, the NanoDrop cannot reliably measure the concentration in impure samples from chemically complex plant extractions, giving an inflated reading. However, as most laboratories do not have easy access or funds to use a Bioanalyzer, the use of the NanoDrop (or Qubit, Life Technologies) is still a laboratory necessity. We advise that using spectrophotomic methods such as NanoDrop or fluorescent-dye methods (e.g., Qubit) are rough and easy ways to assess if the samples are “on track,” but should not be used as an absolute measure for concentration and cannot give a reading of the quality of the ribosomal RNA subunits to prepare for transcriptome sequencing. Low-quality RNA can still be sequenced, but the resulting sequences will be poor, and only determined so after the transcriptome is assembled (R. Cronn, personal communication).

RNA stability: Storing and shipping pure RNA

As with most molecular biological materials, a freeze/thaw process can damage a sample. We found that RNA samples that were not digested immediately with DNase could be stored at 4°C for 2 to 3 d before digestion with no apparent change in quality (data not shown). Once the DNA was digested, a 3-μL aliquot was run on the Agilent 2100 Bioanalyzer, and the remaining samples were placed at −80°C for storage until it was mailed. The pure RNA was sent in the mail for library construction and sequencing after drying down onto specially coated tubes (i.e., GenVault, now renamed as GenTegra; IntegenX, Pleasanton, California, USA) that inhibit RNase activity and stabilize the RNA at room temperature. In general, samples extracted with Options 1 and 2 were more likely to make it through storage, GenVault shipping, and resuspension at BGI than were samples extracted with Option 3. The Option 3 samples were often degraded beyond use upon resuspension at the BGI sequencing facility (Fig. 2A, B and Appendix 2 [gray cells]).

CONCLUSIONS

Use of the TRIzol supplemented with sarkosyl followed by removal of DNA with the TURBO DNA-free kit (Option 1) is an efficient and effective means of extracting RNA from a diverse array of plants, especially those that are woody, aromatic, or aquatic. With the addition of the traditional CTAB method prior to the TRIzol (Option 2), even the most stubborn taxa were mostly successful and gave consistent RNA quality measures. Option 3, which has been used successfully in many laboratories (including our own, e.g., Buggs et al., 2009; Johnson et al., 2012) for RNA isolation, is not the most efficient or robust method for obtaining high-quantity and -quality RNA in transcriptomics across the Embryophyta. Despite the success of the protocols described here, our methods were not successful for some plants that contain high amounts of mucilage, such as Opuntia sp.

Click here for additional data file.

Appendix 1.

List of taxa from which RNA was extracted. Voucher information for the accessions includes the collector, collection number, herbarium acronym, collection location, and georeferenced coordinates of the collection site.

Taxon	Family	Voucher ID	Herbarium	Collection information	Geographic coordinates
Agrimonia eupatoria L.	Rosaceae	Chase 38775	K	RBG Kew, Living Collection	51.47, 0.295
Alluaudiopsis marnieriana Rauh	Didiereaceae	Soltis and Miles 2981	FLAS	U of Florida greenhouse	29.64, −82.34
Alnus serrulata (Aiton) Willd.	Betulaceae	Soltis and Miles 2964	FLAS	U of Florida campus	29.64, −82.34
Amelanchier canadensis (L.) Medik.	Rosaceae	Chase 38778	K	RBG Kew, Living Collection	51.47, 0.295
Anisacanthus quadrifidus (Vahl) Nees	Acanthaceae	Soltis and Miles 2970	FLAS	U of Florida campus	29.64, −82.34
Antirrhinum majus L.	Plantaginaceae	Soltis and Miles 2963	FLAS	U of Florida campus	29.65, −82.34
Aristida stricta Michx.	Poaceae	Jackie Rice JDR10	FLAS	Morningside Nature Center, Gainesville, FL	29.65, −82.27
Aruncus dioicus (Walter) Fernald	Rosaceae	Chase 38772	K	RBG Kew, Living Collection	51.47, 0.295
Astilbe chinensis (Maxim.) Franch. & Sav.	Saxifragaceae	Chase 38784	K	RBG Kew, Living Collection	51.47, 0.295
Bacopa caroliniana (Walter) B. L. Rob.	Plantaginaceae	Soltis and Miles 2974	FLAS	U of Florida greenhouse	29.64, −82.34
Bischofia javanica Blume	Phyllanthaceae	Soltis and Miles 2978	FLAS	U of Florida greenhouse	29.64, −82.34
Bixa orellana L.	Bixaceae	Soltis and Miles 2985	FLAS	U of Florida greenhouse	29.64, −82.34
Boykinia jamesii var. heucheriformis (Rydb.) Engl.	Saxifragaceae	Chase 38782	K	RBG Kew, Living Collection	51.47, 0.295
Canella winterana (L.) Gaertn.	Canellaceae	Soltis and Miles 2995	FLAS	U of Florida greenhouse	29.64, −82.34
Castanea pumila (L.) Mill.	Fagaceae	Soltis and Miles 2977	FLAS	U of Florida campus	29.64, −82.34
Ceratopteris sp. Brongn.	Pteridaceae	Soltis and Miles 3005	FLAS	U of Florida greenhouse	29.64, −82.34
Cercocarpus ledifolius Nutt.	Rosaceae	Chase 38780	K	RBG Kew, Living Collection	51.47, 0.295
Cicerbita plumieri Kirschl.	Asteraceae	Edward Schilling 11-48	TENN	U of Tennessee campus	35.95, −83.92
Corynocarpus laevigatus J. R. Forst. & G. Forst.	Corynocarpaceae	Soltis and Miles 2986	FLAS	U of Florida greenhouse	29.64, −82.34
Cotoneaster transcaucasicus Pojark.	Rosaceae	Chase 38779	K	RBG Kew, Living Collection	51.47, 0.295
Cryptocarya alba (Molina) Looser	Lauraceae	Soltis and Miles 2998	FLAS	U of Florida greenhouse	29.64, −82.34
Deutzia scabra Thunb.	Hydrangeaceae	Soltis and Miles 2965	FLAS	U of Florida campus	29.64, −82.34
Disporopsis pernyi (Hua) Diels	Asparagaceae	Mike Heaney JMH2546	FLAS	U of Florida greenhouse	29.64, −82.34
Dryas octopetala L.	Rosaceae	Chase 38781	K	RBG Kew, Living Collection	51.47, 0.295
Epifagus virginiana (L.) W. P. C. Barton	Orobanchaceae	Soltis and Miles 2996 (Paul Manos 1800)	DUKE	Duke University campus	36.00, −78.94
Forestiera segregata (Jacq.) Krug & Urb.	Oleaceae	Soltis and Miles 2969	FLAS	U of Florida campus	29.64, −82.34
Frullania sp. Raddi	Jubulaceae	Von Konrat 10021	F	Chile, NW shore of Isla Londonderry, SW arm of Bahia Isabel	−54.98, −70.87
Geum quellyon Sweet	Rosaceae	Chase 38770	K	RBG Kew, Living Collection	51.47, 0.295
Gunnera chilensis Lam.	Gunneraceae	Soltis and Miles 2966	FLAS	U of Florida greenhouse	29.64, −82.34
Gunnera manicata Linden ex André	Gunneraceae	Soltis and Miles 2938	FLAS	U of Florida greenhouse	29.64, −82.34
Hilleria latifolia (Lam.) H. Walter	Phytolaccaceae	Soltis and Miles 2976	FLAS	U of Florida campus	29.64, −82.34
Hydrocotyle umbellata L.	Araliaceae	Soltis and Miles 2959	FLAS	U of Florida campus	29.64, −82.34
Illicium floridanum J. Ellis	Schisandraceae	Soltis and Miles 2960	FLAS	Gainesville, FL, home garden; Soltis	29.67, −82.36
Kerria japonica (L.) DC.	Rosaceae	Chase 38777	K	RBG Kew, Living Collection	51.47, 0.295
Kigelia africana (Lam.) Benth.	Bignoniaceae	Soltis and Miles 2992	FLAS	U of Florida greenhouse	29.64, −82.34
Kirkia wilmsii Engl.	Kirkiaceae	Soltis and Miles 2982	FLAS	U of Florida greenhouse	29.64, −82.34
Krameria lanceolata Torr.	Krameriaceae	Soltis and Miles 2991	FLAS	Rte. 24, 0.5 mi. W of Alachua Co. line; N side of rd.; sandhill	29.46, −82.61
Lachnanthes caroliniana (Lam.) Dandy	Haemodoraceae	Soltis and Miles 2988	FLAS	Rt 24, N of roadside, 10 mi W of Alachua Co. line	29.41, −82.67
Lactuca graminifolia Michx.	Asteraceae	Edward Schilling LG-1	TENN	U of Tennessee campus	35.95, −83.92
Lagerstroemia indica L.	Lythraceae	Soltis and Miles 2971	FLAS	U of Florida campus	29.64, −82.34
Licania michauxii Prance	Chrysobalanaceae	Soltis and Miles 2990	FLAS	Gainesville, FL, home garden; Judd	29.57, −82.42
Lindera benzoin (L.) Blume	Lauraceae	Soltis and Miles 2968	FLAS	U of Florida campus	29.64, −82.34
Malus baccata (L). Borkh. var. jackii Borkh.	Rosaceae	Chase 38773	K	RBG Kew, Living Collection	51.47, 0.295
Mammea americana L.	Calophyllaceae	Soltis and Miles 3003	FLAS	U of Florida greenhouse	29.64, −82.34
Melia azedarach L.	Meliaceae	Soltis and Miles 2961	FLAS	U of Florida campus	29.64, −82.35
Michelia maudiae Dunn	Magnoliaceae	Soltis and Miles 2954	FLAS	U of Florida campus	29.64, −82.34
Micranthes geum (L.) Small	Saxifragaceae	Chase 38790	K	RBG Kew, Living Collection	51.47, 0.295
Microtea debilis Sw.	Phytolaccaceae	Soltis and Miles 2997	FLAS	University of Cambridge campus	52.20, −0.116
Muntingia calabura L.	Muntingiaceae	Soltis and Miles 2984	FLAS	U of Florida greenhouse	29.64, −82.34
Myrica pumila (Michx.) Small	Myricaceae	Jackie Rice JDR9	FLAS	Morningside Nature Center, Gainesville, FL	29.65, −82.27
Nandina domestica Thunb.	Berberidaceae	Soltis and Miles 2972	FLAS	U of Florida campus	29.64, −82.34
Nolina bigelovii (Torr.) S. Watson	Asparagaceae	Mike Heaney JMH2984	FLAS	U of Florida greenhouse	29.64, −82.34
Opuntia austrina Small	Cactaceae	Lucas Majure LCM_3450	FLAS	U of Florida greenhouse	29.64, −82.34
Opuntia pusilla (Haw.) Haw.	Cactaceae	Lucas Majure LCM_753	FLAS	U of Florida greenhouse	29.64, −82.34
Peliosanthes minor Yamam.	Asparagaceae	Mike Heaney JMH2549	FLAS	U of Florida greenhouse	29.64, −82.34
Peltoboykinia watanabei (Yatabe) H. Hara	Saxifragaceae	Chase 38787	K	RBG Kew, Living Collection	51.47, 0.295
Persea borbonia (L.) Spreng.	Lauraceae	Soltis and Miles 2980	FLAS	U of Florida campus	29.64, −82.34
Philadelphus inodorus L.	Hydrangeaceae	Soltis and Miles 2953	FLAS	U of Florida campus	29.64, −82.34
Phoradendron leucarpum (Raf.) Reveal & M. C. Johnst.	Santalaceae	Soltis and Miles 2957	FLAS	U of Florida campus	29.64, −82.34
Physocarpus opulifolius (L.) Maxim.	Rosaceae	Chase 38776	K	RBG Kew, Living Collection	51.47, 0.295
Podostemum sp. Michx.	Podostemaceae	Soltis and Miles 2994	FLAS	Massachusetts	NA
Pogostemon sp. Desf.	Lamiaceae	Grant Godden GGT4	FLAS	Gainesville, FL, home garden; Godden	29.65, −82.31
Poliomintha bustamanta B. L. Turner	Lamiaceae	Grant Godden GGT1	FLAS	Gainesville, FL, home garden; Godden	29.65, −82.31
Polypremum procumbens L.	Tetrachondraceae	Soltis and Miles 2989	FLAS	Gainesville, FL, home garden; Judd	29.57, −82.42
Prunus prostrata Labill.	Rosaceae	Chase 38785	K	RBG Kew, Living Collection	51.47, 0.295
Pteris ensiformis Burm. f.	Pteridaceae	Soltis and Miles 3001	FLAS	U of Florida greenhouse	29.64, −82.34
Punica granatum L.	Lythraceae	Soltis and Miles 2973	FLAS	U of Florida campus	29.64, −82.34
Pyrus calleryana Decne.	Rosaceae	Chase 38791	K	RBG Kew, Living Collection	51.47, 0.295
Rhamnus caroliniana Walter	Rhamnaceae	Soltis and Miles 2952	FLAS	U of Florida campus	29.64, −82.34
Rivina humilis L.	Phytolaccaceae	Soltis and Miles 2975	FLAS	U of Florida greenhouse	29.64, −82.34
Rodgersia podophylla A. Gray	Saxifragaceae	Chase 38783	K	RBG Kew, Living Collection	51.47, 0.295
Ruellia brittoniana Leonard	Acanthaceae	Soltis and Miles 2962	FLAS	Gainesville, FL, home garden; Soltis	29.67, −82.36
Salvinia sp. Ség.	Salviniaceae	Soltis and IEJ-T 3013	FLAS	U of Florida greenhouse	29.64, −82.34
Sambucus canadensis L.	Adoxaceae	Soltis and Miles 2955	FLAS	U of Florida campus	29.64, −82.34
Sanguisorba minor Scop.	Rosaceae	Chase 38771	K	RBG Kew, Living Collection	51.47, 0.295
Saxifraga geum L. var. gracilis	Saxifragaceae	Chase 38790	K	RBG Kew, Living Collection	51.47, 0.295
Schlegelia parasitica (Sw.) Miers ex Griseb.	Schlegeliaceae	Soltis and Miles 2983	FLAS	U of Florida greenhouse	29.64, −82.34
Sorbus koehneana C. K. Schneid.	Rosaceae	Chase 38774	K	RBG Kew, Living Collection	51.47, 0.295
Sprekelia formosissima (L.) Herb.	Amaryllidaceae	Garcia 4381	K	U of Florida greenhouse	29.64, −82.34
Strobilanthes dyerianus	Acanthaceae	Soltis and Miles 2958	FLAS	U of Florida greenhouse	29.64, −82.34
Talinum sp. Adans.	Talinaceae	Soltis and Miles 2979	FLAS	U of Florida greenhouse	29.64, −82.34
Tellima breviflora Rydb.	Saxifragaceae	Chase 38789	K	RBG Kew, Living Collection	51.47, 0.295
Teucrium chamaedrys L.	Lamiaceae	Grant Godden GGT3	FLAS	Gainesville, FL, home garden; Godden	29.65, −82.31
Thymus vulgaris L.	Lamiaceae	Grant Godden GGT2	FLAS	Gainesville, FL, home garden; Godden	29.65, −82.31
Tiarella polyphylla D. Don	Saxifragaceae	Chase 38786	K	RBG Kew, Living Collection	51.47, 0.295
Tragopogon castellanus Levier	Asteraceae	Soltis and Miles 2993	FLAS	U of Florida greenhouse	29.64, −82.34
Traubia modesta Ravenna	Amaryllidaceae	Garcia 3014	FLAS	U of Florida greenhouse	29.64, −82.34
Uniola paniculata L.	Poaceae	Richard Hodel RGJH1001	FLAS	St. Augustine Beach, FL	29.86, −81.26
Utricularia sp. L.	Lentibulariaceae	Soltis and Miles 2987	FLAS	U of Florida greenhouse	29.64, −82.34
Zephyranthes citrina Baker	Amaryllidaceae	Garcia 4376	FLAS	U of Florida greenhouse	29.64, −82.34
Ziziphus jujuba Mill.	Rhamnaceae	Soltis and Miles 2956	FLAS	U of Florida campus	29.64, −82.34

Note: NA = not available.

All garden-grown individuals in this study are from unknown seed or stock source and cannot be traced to the possible wild collection site as they are either established landscape plants or part of a permanent greenhouse collection with the main purpose of teaching botany.

Appendix 2.

List of taxa from which RNA was extracted, the method used, and the Agilent 2100 Bioanalyzer quality indicators of the final extraction that was sequenced.

		Option 1			Option 2			Option 3
Family	Taxon	Material isolated (μg)	RINb	rRNA Ratio 26S/18S	Material isolated (μg)	RINb	rRNA Ratio 26S/18S	Material isolated (μg)	RINb	rRNA Ratio 26S/18S	Sequenced?	Voucher ID
Acanthaceae	Anisacanthus quadrifidus  (Vahl) Nees	40	6.4	Missing	—	—	—	46.1	3.9	1.2	Option 1	Soltis and Miles 2970
Acanthaceae	Ruellia brittoniana  Leonard	43	7.6	Missing	—	—	—	29.2	2.4	0	Option 1	Soltis and Miles 2962
Acanthaceae	Strobilanthes  dyerianus	—	—	—	—	—	—	25*	4.9	2.7	Option 3	Soltis and Miles 2958
Adoxaceae	Sambucus  canadensis L.	45.5	4.2	1.4	—	—	—	—	—	—	Option 1	Soltis and Miles 2955
Amaryllidaceae	Sprekelia formosissima  (L.) Herb.	—	—	—	22.1	3.2	0.2	—	—	—	Library construction  failed	Garcia 4381
Amaryllidaceae	Traubia modesta  Ravenna	—	—	—	40	6	1.6	—	—	—	Option 2	Garcia 3014
Amaryllidaceae	Zephyranthes citrina  Baker	—	—	—	22	4.5	0.3	—	—	—	Library construction  failed	Garcia 4376
Araliaceae	Hydrocotyle  umbellata L.	—	—	—	—	—	—	30*	5.9	1.1	Option 3	Soltis and Miles 2959
Asparagaceae	Disporopsis pernyi (Hua)  Diels	33.7	7.2	1.4	—	—	—	—	—	—	Option 1	Mike Heaney JMH2546
Asparagaceae	Nolina bigelovii  (Torr.) S. Watson	33.45	7.9	1.5	—	—	—	—	—	—	Option 1	Mike Heaney JMH2984
Asparagaceae	Peliosanthes minor  Yamam.	21.1	6.9	1.2	—	—	—	—	—	—	Option 1	Mike Heaney JMH2549
Asteraceae	Cicerbita plumieri  Kirschl.	21	7.6	2.5	—	—	—	—	—	—	Option 1	Edward Schilling 11-48
Asteraceae	Lactuca graminifolia  Michx.	54.85	7.6	2.4	—	—	—	—	—	—	Option 1	Edward Schilling LG-1
Asteraceae	Tragopogon  castellanus Levier	19.3	6.3	1	—	—	—	36.7	2.1	0	Option 1	Soltis and Miles 2993
Berberidaceae	Nandina domestica  Thunb.	—	—	—	—	—	—	30*	5.7	1	Option 3	Soltis and Miles 2972
Betulaceae	Alnus serrulata  (Aiton) Willd.	—	—	—	—	—	—	20*	8.2	2.2	Option 3	Soltis and Miles 2964
Bignoniaceae	Kigelia africana  (Lam.) Benth.	250.9	5.1	2.8	—	—	—	—	—	—	Option 1	Soltis and Miles 2992
Bixaceae	Bixa orellana L.	—	—	—	—	—	—	30*	4.8	1.6	Option 3	Soltis and Miles 2985
Cactaceae	Opuntia austrina Small	—	—	—	7.7	8.2	1.4	—	—	—	Low conc.; library  not tried	Lucas Majure LCM_3450
Cactaceae	Opuntia pusilla  (Haw.) Haw.	Tried but failed	Tried but failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	All extractions  failed	Lucas Majure LCM_753
Calophyllaceae	Mammea americana L.	—	—	—	50	6	1.6	—	—	—	Option 2	Soltis and Miles 3003
Canellaceae	Canella winterana  (L.) Gaertn.	42.0	6.3	1.1	—	—	—	28.5	2.6	0.5	Option 1	Soltis and Miles 2995
Chrysobalanaceae	Licania michauxii  Prance	—	—	—	22	7	2	—	—	—	Option 2	Soltis and Miles 2990
Corynocarpaceae	Corynocarpus laevigatus  J. R. Forst. & G. Forst.	126.8	7.3	Missing	—	—	—	42.2	3.4	0.8	Option 1	Soltis and Miles 2986
Didiereaceae	Alluaudiopsis  marnieriana Rauh	15.9	5.9	Missing	—	—	—	27.7	3.3	0.8	Option 1	Soltis and Miles 2981
Fagaceae	Castanea pumila  (L.) Mill.	—	—	—	—	—	—	25*	6.5	1.5	Option 3	Soltis and Miles 2977
Gunneraceae	Gunnera chilensis Lam.	—	—	—	—	—	—	25*	6.7	1.6	Option 3	Soltis and Miles 2966
Gunneraceae	Gunnera manicata  Linden ex André	—	—	—	—	—	—	81	5.6	1	Option 3	Soltis and Miles 2938
Haemodoraceae	Lachnanthes  caroliniana (Lam.) Dandy	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	All extractions  failed	Soltis and Miles 2988
Hydrangeaceae	Deutzia scabra Thunb.	—	—	—	1.75	2.6	0	82.3	2.9	0.5	Option 3	Soltis and Miles 2965
Hydrangeaceae	Deutzia scabra Thunb.										Low conc.; library  not tried	Soltis and Miles 2965
Hydrangeaceae	Philadelphus  inodorus L.	—	—	—	—	—	—	35*	4.9	1.1	Option 3	Soltis and Miles 2953
Jubulaceae	Frullania sp. Raddi	—	—	—	40	6.1	1.6	—	—	—	Option 2	Von Konrat 10021
Kirkiaceae	Kirkia wilmsii Engl.	—	—	—	—	—	—	13.8*	6.9	0.7	Option 3	Soltis and Miles 2982
Krameriaceae	Krameria lanceolata  Torr.	—	—	—	—	—	—	23.8	4.5	1.1	Option 3	Soltis and Miles 2991
Lamiaceae	Pogostemon sp. Desf.	39.35	7.5	1.6	—	—	—	—	—	—	Option 1	Grant Godden GGT4
Lamiaceae	Poliomintha bustamanta  B. L. Turner	—	—	—	46	6	1.6	—	—	—	Option 2	Grant Godden GGT1
Lamiaceae	Teucrium chamaedrys L.	72.85	7.4	1.7	—	—	—	—	—	—	Option 1	Grant Godden GGT3
Lamiaceae	Thymus vulgaris L.	71.1	7.5	1.4	—	—	—	—	—	—	Option 1	Grant Godden GGT2
Lauraceae	Cryptocarya alba  (Molina) Looser	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but  failed	Tried but failed	All extractions failed	Soltis and Miles 2998
Lauraceae	Lindera benzoin  (L.) Blume	2.7	2.6	0	48.5	6	1.6	27.8	2.7	0.2	Option 2	Soltis and Miles 2968
Lauraceae	Persea borbonia  (L.) Spreng.	—	—	—	—	—	—	18*	5.3	1.2	Option 3	Soltis and Miles 2980
Lentibulariaceae	Utricularia sp. L.	—	—	—	—	—	—	30.9	4.3	1.2	Option 3	Soltis and Miles 2987
Lythraceae	Lagerstroemia indica L.	—	—	—	—	—	—	7	8	1.8	Option 3	Soltis and Miles 2971
Lythraceae	Punica granatum L.	—	—	—	—	—	—	20*	7.5	1.9	Option 3	Soltis and Miles 2973
Magnoliaceae	Michelia maudiae Dunn	—	—	—	—	—	—	28.5*	4.1	0.6	Option 3	Soltis and Miles 2954
Malvaceae	Grewia occidentalis							14.04	3.2	1	Degraded	Missing
Meliaceae	Melia azedarach L.	202	6.5	1.5	—	—	—	—	—	—	Option 1	Soltis and Miles 2961
Muntingiaceae	Muntingia calabura L.	—	—	—	21	8.1	1.7	122.8	2.9	0.3	Option 2	Soltis and Miles 2984
Myricaceae	Myrica pumila  (Michx.) Small	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	14.5	3.1	1.3	All extractions  failed	Jackie Rice JDR9
Oleaceae	Forestiera segregata  (Jacq.) Krug & Urb.	27.0	6.5	Missing	—	—	—	24.5	3.0	0.5	Option 1	Soltis and Miles 2969
Orobanchaceae	Epifagus virginiana  (L.) W. P. C. Barton	10.9	3.1	0.6	38.3	7.2	1.6	46.5	2.1	0	Option 2	Soltis and Miles 2996 (Paul Manos 1800)
Phyllanthaceae	Bischofia javanica  Blume	—	—	—	—	—	—	10*	7.8	3	Option 3	Soltis and Miles 2978
Phytolaccaceae	Hilleria latifolia  (Lam.) H. Walter	24.1	5.8	0.5	—	—	—	35.6	2.7	0.1	Option 1	Soltis and Miles 2976
Phytolaccaceae	Microtea debilis Sw.	43.9	6.3	1	—	—	—	29.4	3.0	0.4	Option 1	Soltis and Miles 2997
Phytolaccaceae	Rivina humilis L.	19.45	6.6	1	—	—	—	28.42	2.8	0.4	Option 1	Soltis and Miles 2975
Plantaginaceae	Antirrhinum majus L.	64.0	5.4	0.5	—	—	—	50.9	4.1	0.8	Option 1	Soltis and Miles 2963
Plantaginaceae	Bacopa caroliniana  (Walter) B. L. Rob.	26.0	7.8	1.4	—	—	—	28.4	3.5	1.5	Option 1	Soltis and Miles 2974
Poaceae	Aristida stricta Michx.	54.5	5.6	Missing	—	—	—	—	—	—	Option 1	Jackie Rice JDR10
Poaceae	Uniola paniculata L.	35.85	7.6	2.5	—	—	—	—	—	—	Option 1	Richard Hodel RGJH1001
Podostemaceae	Podostemum sp. Michx.	—	—	—	23.9	4.7	0.8	44.7	2.1	0	Library construction  failed	Soltis and Miles 2994
Pteridaceae	Ceratopteris sp. Brongn.	—	—	—	Missing	Missing	Missing	—	—	—	Option 2	Soltis and Miles 3005
Pteridaceae	Pteris ensiformis Burm. f.	28	8.4	1.6	—	—	—	—	—	—	Option 1	Soltis and Miles 3001
Rhamnaceae	Rhamnus caroliniana  Walter	162	5.8	1.1	—	—	—	80	3.1	0.5	Option 1	Soltis and Miles 2952
Rhamnaceae	Ziziphus jujuba Mill.	0.15	2.7	0	45	6.5	1.6	25*	3.6	0.6	Option 2	Soltis and Miles 2956
Rosaceae	Agrimonia eupatoria L.	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Chase 38775
Rosaceae	Amelanchier canadensis  (L.) Medik.	—	—	—	50	7	1.7	—	—	—	Option 2	Chase 38778
Rosaceae	Aruncus dioicus  (Walter) Fernald	—	—	—	122	6.4	1.6	—	—	—	Option 2	Chase 38772
Rosaceae	Cercocarpus ledifolius  Nutt.	—	—	—	40	7.1	2.5	—	—	—	Option 2	Chase 38780
Rosaceae	Cotoneaster transcaucasicus Pojark.	—	—	—	154	8.2	1.5	—	—	—	Option 2	Chase 38779
Rosaceae	Dryas octopetala L.	—	—	—	67	7.3	1.6	—	—	—	Option 2	Chase 38781
Rosaceae	Geum quellyon Sweet	—	—	—	78.2	7.7	1.6	—	—	—	Option 2	Chase 38770
Rosaceae	Kerria japonica (L.) DC.	—	—	—	260	8.4	1.8	—	—	—	Option 2	Chase 38777
Rosaceae	Malus baccata (L). Borkh. var. jackii Borkh.	—	—	—	75	6.7	1.9	—	—	—	Option 2	Chase 38773
Rosaceae	Physocarpus opulifolius  (L.) Maxim.	—	—	—	163	8.3	2.2	—	—	—	Option 2	Chase 38776
Rosaceae	Prunus prostrata Labill.	—	—	—	155.6	7.1	1.7	—	—	—	Option 2	Chase 38785
Rosaceae	Pyrus calleryana Decne.	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Chase 38791
Rosaceae	Sanguisorba minor Scop.	—	—	—	239.8	8.2	1.6	—	—	—	Option 2	Chase 38771
Rosaceae	Sorbus koehneana  C. K. Schneid.	—	—	—	38.7	6.6	1.3	—	—	—	Option 2	Chase 38774
Salviniaceae	Salvinia sp. Ség.	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Soltis and IEJ-T 3013
Santalaceae	Phoradendron leucarpum  (Raf.) Reveal & M. C. Johnst.	—	—	—	37	4.1	0.3	59.8	2.3	0	Option 2	Soltis and Miles 2957
Saxifragaceae	Astilbe chinensis  (Maxim.) Franch. & Sav.	—	—	—	60	7	1.6	—	—	—	Option 2	Chase 38784
Saxifragaceae	Boykinia jamesii var.  heucheriformis (Rydb.) Engl.	—	—	—	63.9	6	2.1	—	—	—	Option 2	Chase 38782
Saxifragaceae	Micranthes geum  (L.) Small	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Chase 38790
Saxifragaceae	Peltoboykinia watanabei  (Yatabe) H. Hara	—	—	—	70	7	1.7	—	—	—	Option 2	Chase 38787
Saxifragaceae	Rodgersia podophylla  A. Gray	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Chase 38783
Saxifragaceae	Saxifraga geum L. var.  gracilis	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	Tried but failed	All extractions  failed	Chase 38790
Saxifragaceae	Tellima breviflora Rydb.	—	—	—	41.5	7.1	1.5	—	—	—	Option 2	Chase 38789
Saxifragaceae	Tiarella polyphylla  D. Don	—	—	—	30	7.8	1.6	—	—	—	Option 2	Chase 38786
Schisandraceae	Illicium floridanum  J. Ellis	—	—	—	—	—	—	25*	4.8	0.8	Option 3	Soltis and Miles 2960
Schlegeliaceae	Schlegelia parasitica  (Sw.) Miers ex Griseb.	18.2	8.2	1.5	—	—	—	7.8	2.1	0	Option 1	Soltis and Miles 2983
Talinaceae	Talinum sp. Adans.	8.6	7.3	Missing	—	—	—	15.4	2.3	0	Option 1	Soltis and Miles 2979
Tetrachondraceae	Polypremum  procumbens L.	28.3	6.8	1.5	—	—	—	48.8	2.8	0.5	Yes	Soltis and Miles 2989

Note: — = method was not tried on the species.

Gray cells are those with degraded RNA and either the library was not attempted or it failed. All samples were extracted from 60 to 100 mg of plant leaf tissue (for aquatics and succulents, 200 mg). The taxa that failed with all attempts were: Agrimonia eupatoria L. (Rosaceae), Cryptocarya alba (Molina) Looser (Lauraceae), Lachnanthes caroliniana (Lam.) Dandy (Haemodoraceae), Micranthes geum (L.) Small (Saxifragaceae), Opuntia pusilla (Haw.) Haw. (Cactaceae), Pyrus calleryana Decne. (Rosaceae), Rodgersia podophylla A. Gray (Saxifragaceae), Salvinia sp. Ség. (Salviniaceae), and Saxifraga geum L. var. gracilis (Saxifragaceae). Failed library construction despite having satisfactory metrics: Sprekelia formosissima (L.) Herb. (Amaryllidaceae) and Zephyranthes citrina Baker (Zephranthaceae).

RIN (RNA integrity number) is a measure from the Agilent 2100 Bioanalyzer that combines concentration and expected presence of subunits.

Marks the samples where an isolate was extracted from two to four extractions and pooled to be sequenced. (Not all extractions in Table 1 are in this table as not all were ran on the Bioanalyzer.)