To test our proposed protocol (hereafter named SILEX, for SILica matrix EXtraction), leaf and fruit tissue from non-recalcitrant species and leaf tissue from recalcitrant species was sampled for four different trials. In a first trial, leaf tissue from a total of 1,860 accessions of tomato (S. lycopersicum) and its wild relatives was extracted to compare the quality, quantity and integrity of DNA extracted using SILEX and the commercial kit sbeadex maxi plant kit (hereafter SMP kit; LGC Genomics, Teddington, UK) for SPET genotyping. Extractions were performed on different days over several months
In a second trial, in order to evaluate the appropriateness of SILEX in different plant tissues, 50 mg of fresh and 30 mg of lyophilized fruit tissue of tomato, eggplant (S. melongena) and pepper (Capsicum annuum) were extracted. The fruit tissue was collected, immediately frozen in liquid N2 and lyophilized In a third trial, the suitability of SILEX for DNA extraction in recalcitrant species was assessed using leaves tissue of six species, cassava (Manihot esculenta), grapevine (Vitis vinifera), loquat (Eriobotrya japonica), banana (Musa × paradisiaca), naranjillo (Solanum bonariense), and strawberry (Fragaria × ananassa), selected to represent a wide range of recalcitrant species presenting different contaminants and secondary metabolites that interfere with DNA extraction. Extractions from recalcitrant plants made by SILEX were compared with those carried out using the standard CTAB protocol  and the commercial SMP kit following the manufacturer's instructions. Finally, the suitability of SILEX to extract clean and high-molecular-weight DNA for third-generation sequencing was assessed in the silverleaf nightshade (S. elaeagnifolium), a wild relative of eggplant , that we selected for the difficulty to obtain contaminant-free DNA due to its high content in phenolics .
Solutions, reagents and consumables
- 2 ml Sarstedt Microtube (sarstedt.com, Cat No. 72.691)
- 5 mm Glass beads (www.vwr.com, Cat No. MARI4901005)
- N-Cetyl-N,N,N-trimethylammonium bromide, CTAB (itwreagents.com, Cat No. A6284.0500)
- Polyvinylpyrrolidone, PVP-40 (www.sigmaaldrich.com, Cat No. PVP40-500G)
- Ethylenediaminetetraacetic acid, EDTA (itwreagents.com, Cat No. 131026.1210)
- Trizma® hydrochloride, Tris HCl (www.sigmaaldrich.com, Cat No. 93363-500G)
- Sodium Chloride (itwreagents.com, Cat No. 131659.1211)
- β-Mercaptoethanol (www.sigmaaldrich.com, Cat No. M6250-100ML)
- RNase A (www.vwr.com, Cat No. NA-03)
- Chloroform Essent® (scharlab.com, Cat No. CL1981000)
- Isoamyl alcohol Essent® (scharlab.com, Cat No. AL02851000)
- Polyethylene Glycol 8000 (itwreagents.com, Cat No. 146224.1211)
- Absolute Ethanol ExpertQ® (www.scharlab.com, Cat No. ET0002025P)
- Silicon dioxide (www.fishersci.se, Cat No. S5631-500G)
- Hydrochloric acid (sigmaaldrich.com, Cat No. H1758-500ML)
- Absolute Ethanol ExpertQ® (www.scharlab.com, Cat No. ET0002025P)
- Tris Base (itwreagents.com, Cat No. 14194.1211)
- Qiagen TissueLyser II (www.qiagen.com, Cat No. 85300)
- Thermoblock (www.vwr.com, Cat No. 12621-096)
- Eppendorf Centrifuge 5424 (eppendorf.com, Cat. No. 5404000010)
Extraction buffer: 2% (w/v) CTAB, 2% (w/v) PVP-40, 20 mM EDTA, 100 mM Tris HCl (pH 8.0) and 1.40 M NaCl. The buffer may be stored for several months at room temperature.
Protein precipitation buffer: 24 parts of chloroform and 1 part of isoamyl alcohol. It may be stored for several months at room temperature.
Binding buffer: 2.5 M NaCl and 20% PEG 8000. It may be stored for several months at room temperature.
Silica matrix buffer: Mix 5 g of silicon dioxide with 50 ml of MilliQ water and let stand for 24 hours. Discard the supernatant and resuspend the pellet in 50 ml of MilliQ water and let stand for another 5 hours. Discard the supernatant and resuspend the pellet in 1:1 (v/v) MilliQ water. Finally, add 10 µl of HCl 36% per ml of silica matrix solution obtained. It may be stored for several months at room temperature.
Washing buffer: Fresh prepared ethanol 70%. It may be stored for several months at room temperature.
Elution buffer: 10 mM Tris HCl (pH 8.0) and 1 mM EDTA (pH 8.0). It may be stored for several months at room temperature.
SILEX DNA extraction protocol
- Add a 5 mm glass bead to a 2 ml tube, place around 50 mg of fresh or lyophilized tissue into the tube and immediately frozen in liquid nitrogen.
- Place the tubes in the Qiagen TissueLyser adapters, grind the samples for 60 secs at 13,000 rpm and immediately return the samples in liquid nitrogen.
NOTE: This is a critical step and can greatly influences the final DNA recovery. Avoid sample thaw and pre-chill Qiagen TissueLyser plate adapters. Check that the plant material has become a fine powder.
- Take the tube from the liquid nitrogen and gently tap it vertically to settle the plant material at the bottom of the tube. Add 1 ml of extraction buffer, add 14 µl of β-mercaptoethanol and gently mix the tube until complete homogenization. Then, add 2 µl of RNase (10 mg/ml) and incubate in a thermoblock for 30 min at 65 °C.
NOTE: Avoid sample thaw before adding the extraction buffer. Preheat the thermoblock at 65 °C.
- Put the samples on ice for 5 min. Add 700 µl of protein precipitation buffer and gently vortex or (for very high molecular weight DNA) gently invert by hand thoroughly for approximately 20 seconds.
- Centrifuge at 11,000 rpm for 5 min at room temperature, carefully recover around 800 µl of the supernatant phase and transfer it to a new 2 ml tube.
NOTE: Pipette carefully to avoid dragging the interphase. Interphase contaminants can largely affect the final quality of the DNA.
- Add 480 μl of binding buffer and gently invert the tube by hand until complete mixing. Subsequently, add 720 μl of absolute ethanol and gently invert the tube again for a few seconds until complete mixing.
NOTE: The amount of binding buffer plus ethanol must be 1.5 volumes of the supernatant recovered in step 5. In the binding buffer plus ethanol mix, 40% should be the binding buffer and 60% should be absolute ethanol.
- Add 20 µl of silica matrix buffer and mix gently during 5 min (by hand or using an orbital shaker).
- Spin down the silica for 5 to 6 seconds and discard the supernatant by decantation.
NOTE: Longer centrifugation times will make it difficult to resuspend the silica in the subsequent steps.
- Add 700 μl of washing buffer and shake gently by hand until a uniform dispersion of the silica is obtained.
- Spin down the silica for 5 to 6 seconds, gently discard the supernatant by decantation and let dry at room temperature for 5 min.
NOTE: Make sure that all ethanol is completely evaporated.
- Add 100 µl of elution buffer, shake gently by hand until the pellet is resuspended and incubate 5 min at 65 ºC.
- Centrifuge at 14,000 rpm for 10 min at room temperature and transfer 90 µl of the supernatant to a new tube.
DNA concentration and quality
DNA integrity was checked by electrophoresis on a 0.8% agarose gel (Condalab, Madrid, Spain) in 1X TAE buffer (GenoChem World, Valencia, Spain) stained with GelRed® (Biotium, Fremont, CA, USA) at a constant voltage of 100 V for 50 min. Gel Doc XR+ System transilluminator (Bio-Rad, Hercules, CA, USA) was used to visualized agarose gels. For high-molecular-weight DNA, the size and integrity were tested by pulse-field gel electrophoresis was run at 3.3 V/cm in 15-second cycles with an angle of 120º for 24 h at 4 ºC with 0.8% agarose in TB buffer.
DNA yield and quality were measured spectrophotometrically using NanoDrop™ ND-1000 (Thermo Scientific, Waltham, MA, USA). A260/A280 and A260/A230 ratios were measured to determine, respectively, protein and polysaccharide contamination. DNA quantity was also quantified with a Qubit™ 2.0. Fluorometer (Thermo Scientific, Waltham, MA, USA). An aliquot of 2 µl of each sample was examined using the Qubit™ dsDNA BR Assay Kit (Thermo Scientific, Waltham, MA, USA) according to the instructions of the manufacturer.
In addition, the concentration of DNA obtained from the 1,860 tomato samples was measured fluorometrically using Quant-iT™ PicoGreen™ dsDNA Assay Kit (hereafter PicoGreen, Thermo Scientific, Waltham, MA, USA) and a 96-wells plate reader VICTOR3 1420 (PerkinElmer, Waltham, MA, USA) equipped with an excitation filter F485 and emission filter F535.
To check the suitability of the DNA extraction method for sequencing applications where DNA is fragmented, approximately 1 µg of DNA was digested for 1 h at 37 ºC followed by 20 min at 65 ºC with restriction enzymes EcoRI (New England Biolabs, Ipswich, MA, USA). The digestion was evaluated through 1% agarose electrophoresis as above.
High-throughput genotyping quality check
For the first trial, sequencing of tomato samples for genotyping by SPET was performed with an Illumina NextSeq 500 platform (Illumina Inc., San Diego, CA, USA), following the manufacturer protocol. Phred values were obtained using FastQC Version 0.11.8. and plotted in R  using the package ggplot2 .
Suitability of extracted DNA for third-generation sequencing platforms
For the third trial, 5 µg of S. elaeagnifolium DNA from a single extraction were size-selected using the Circulomics SRE-XL-Kit (Circulomics Inc., Baltimore, MD, USA). For library preparation, 1 µg of the size-selected DNA was used to prepare each of the three Nanopore LSK-109 libraries. Two of these libraries were sequenced on a MinION R9.4.1 (Oxford Nanopore, Oxford, UK) and the third was loaded on a PromethION PRO-002 (Oxford Nanopore, Oxford, UK). All three sequencing runs were basecalled using Oxford Nanopores Guppy basecaller version 3.2.2 (Oxford Nanopore, Oxford, UK) using the high accuracy basecalling models.