Next-generation sequencing (NGS) underpins recently developed high-throughput molecular marker technologies, such as genotyping-by-sequencing (GBS). These methodologies can identify thousands of informative single nucleotide polymorphism (SNP) markers cost-effectively and rapidly in almost any species, regardless of genomic resources [1]. DNA quality is paramount for sequencing and restriction enzyme-based GBS platforms, which require high molecular weight genomic DNA (gDNA) largely free of contaminants, such as polysaccharides or phenols [2, 3]. These contaminants can inhibit sequencing quality as well as restriction enzyme activity and subsequent procedures, thereby providing a source of inconsistent or unreliable SNP data, which has significant implications for genotype analysis [4].
DNA extraction, particularly from plants, can be problematic due to cell wall and fibre components that interfere with tissue homogenisation, in addition to contamination from phenols and carbohydrates. While many protocols are available to address these challenges [3, 5,6,7], most are expensive (US$6–$9/sample) or relatively low-throughput [3, 8]. Furthermore, many plant extraction methods rely on organic solvents or include compounds such as β-mercaptoethanol [3, 5,6,7] which render protocol automation without a fume hood difficult. Whitlock et al. [2] described a non-organic solvent, high-throughput DNA extraction protocol suitable for PCR-based assays from freeze-dried plant and insect tissue. We have improved and extended this protocol to routinely extract sequencing-quality high molecular weight gDNA from fresh and dried tissue in a high-throughput manner across a wide range of forage, crop, and model species while significantly enhancing cost and time-effectiveness.
Materials
Reagents
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Sodium chloride (www.thermofisher.com, Cat code: BSPSL944.5).
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Tris (www.thermofisher.com, Cat code: AJA180-500 g).
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EDTA (www.thermofisher.com, Cat code: AJA180-500G).
CAUTION HSNO Classes: 6.1E (oral), 6.3B, 6.4A, 9.1C (fish). Hazard statement: H303 May be harmful if swallowed; H316 Causes mild skin irritation; H319 Causes serious eye irritation; H412 Harmful to aquatic life with long lasting effects.
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Sodium sulphite (www.vwr.com, Cat code: 28130.260).
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Sodium dodecyl sulphate (www.vwr.com, Cat code: VWRC442444H).
CAUTION HSNO Classes: 6.1C, 6.1D (oral), 6.3B, 6.4A, 9.1D (algal), 9.1D (crustacean), 9.1D (fish), 9.2D, 9.3C. Hazard statement: H302 Harmful if swallowed, H316 Causes mild skin irritation; H319 Causes serious eye irritation; H413 May cause long lasting harmful effects to aquatic life; H423 Harmful to the soil environment; H433 Harmful to terrestrial vertebrates.
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Potassium acetate (www.sigmaaldrich.com, Cat code: P1147-1 kg).
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Acetic acid (www.merckmillipore.com, Cat code: 1000632500).
CAUTION HSNO Classes: 3.1C, 6.1D, 6.1D (inhalation), 6.1D (oral), 6.9B (inhalation), 8.1A, 8.2B, 8.3A, 9.1D (algal), 9.1D (crustacean), 9.1D (fish), 9.3C. Hazard statement: H226 Flammable liquid and vapour; H290 May be corrosive to metals; H302 Harmful if swallowed; H332 Harmful if inhaled; H314 Causes severe skin burns and eye damage; H318 Causes serious eye damage; H371 May cause damage to organs; H373 May cause damage to organs through prolonged or repeated exposure; H413 May cause long lasting harmful effects to aquatic life; H433 Harmful to terrestrial vertebrates.
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Guanidinium chloride (www.vwr.com, Cat code: ALFAA13543.0B).
CAUTION HSNO Classes: 6.1C, 6.1D (oral), 8.2C, 8.3A, 9.3C. Hazard statement: H302 Harmful if swallowed; H314 Causes severe skin burns and eye damage; H318 Causes serious eye damage; H433 Harmful to terrestrial vertebrates. Guanidinium chloride is a strong protein denaturing agent.
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Absolute ethanol (www.vwr.com, Cat code: VWRC20821.365).
CAUTION HSNO Classes: 3.1B, 6.4A. Hazard statement: H225 Highly flammable liquid and vapour H319 Causes serious eye irritation.
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Liquid nitrogen (www.linde-gas.com, Cat code: 4081536554).
CAUTION Hazard statement: H281 Contains refrigerated gas; may cause cryogenic burns or injury.
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20 µg µl−1 1000 mg Proteinase K Bioline (www.bioline.com, Cat code: BIO-37039).
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Qiagen RNase A 2.5 ml (100 mg ml−1; 7000 units ml−1) (www.qiagen.com, Cat code: QIA19101).
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LabServ Pronalys 2–6 mm self-indicating Silica gel (www.thermofisher.com, Cat code: BSPSL519.5).
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1 Kb Plus Ladder (www.thermofisher.com, Cat code: 10787-018).
Equipment
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Corning® 96 Well Clear Round Bottom 1 ml Polypropylene Block (www.corning.com, Cat code: COR3959).
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5/32 inch 440C Stainless steel balls grinding grade (3.97 mm) (www.glenmills.com, Cat code: 7400-003969-6).
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4titude Thermal bond (www.4ti.co.uk, Cat code: 4ti-0591).
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Qiagen TissueLyser II (www.qiagen.com).
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Hettich Rotanta 460R centrifuge (www.hettichlab.com).
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Axygen® 1.1 ml 96 well Deep well plate, clear (www.corning.com, Cat code: P-DW-11-C).
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Axygen® Sealing Mats (www.corning.com, Cat code: AM-2ML-RD).
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Pall AcroPrep™ Advance 96-Well 1 ml Filter plate (www.pall.com, Cat code: PN8132).
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3/32 inch 440C Stainless steel balls grinding grade (2.38 mm) (www.glenmills.com, Cat code: 7400-002381-6).
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4titude PCR seal (www.4ti.co.uk, Cat code: 4ti-0500).
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2 ml Sarstedt Screw Cap Microtubes (www.sarstedt.com, Cat code: SARS72.694.005).
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¼ inch Ceramic Spheres (www.vwr.com, Cat. No. QBIO6540-034).
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Eppendorf® Microcentrifuge 5415D (www.eppendorf.com).
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Hamilton Microlab® STARlet Liquid Handling Workstation (www.hamiltoncompany.com).
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Nanodrop ND-1000 Spectrophotometer (www.thermofisher.com).
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Qubit® 2.0 Fluorometer (www.thermofisher.com).
Reagent setup
Homogenisation buffer (HB)
500 mM sodium chloride, 100 mM Tris (pH 7.4), 50 mM EDTA, 52 mM sodium sulphite, 0.7% (w/v) sodium dodecyl sulphate. Buffer may be stored for several months at room temperature.
Precipitation buffer (PB)
3.6 M potassium acetate, 2.4 M acetic acid. Buffer may be stored for several months at room temperature.
Binding buffer (BB)
To make up 1000 ml, add 191 g of 2 M guanidinium chloride, make up to 333 ml with TE (Tris EDTA 10:1, pH 8), add 667 ml absolute ethanol. Buffer may be stored for several months at room temperature.
Wash buffer (WB)
To make up 1000 ml, add 4 ml 5 M sodium chloride, 2 ml 1 M Tris–HCL (pH 8), 194 ml water, 800 ml absolute ethanol. Buffer may be stored for several months at room temperature.
Plant material
White clover (Trifolium repens), western clover (T. occidentale), pale clover (T. pallescens), red clover (T. pratense), subterranean clover (T. subterraneum), T. uniflorum, perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), rye (Secale cereale) and lucerne/afalfa (Medicago sativa) were sourced from the Margot Forde Germplasm Centre (MFGC), Grasslands Research Centre, AgResearch, Palmerston North, New Zealand. Plants were grown in planting trays or planter bags in glasshouses on site and leaf or grass pseudostem tissue was harvested for DNA isolation. Leaf tissue from apple (Malus pumila) was sampled from trees growing on the AgResearch Grasslands Research Centre campus. Rice (Oryza sativa) pseudostem and Arabidopsis thaliana leaves were sourced from AgResearch colleagues.
Protocol
Fresh tissue gDNA extraction protocol
Critical step Matching bead size with the appropriate well size and plate strength is important to ensure effective tissue grinding and reduces the likelihood of beads jamming in the base of the well, or the plate shattering. To upscale gDNA extraction, the plate may be filled with samples from a single individual.
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1.
Load 50 mg of fresh tissue and two 5/32 inch (3.97 mm) stainless steel beads into each well of a Corning® 1 ml deep well 96-well plate. Heat-seal with a Thermal Bond seal, which provides greater protection against beads breaking through the seal during the grinding process, and float the plate in liquid nitrogen for 5 min. Similarly, pre-chill Qiagen TissueLyser plate adapters.
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2.
Place the 96-well plate in the Qiagen TissueLyser plate adapter and grind tissue at 30 Hz for 1 min then, for more consistent grinding, repeat after changing the plate orientation in the adaptors. If homogenising only one plate at a time, use a second balance plate.
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3.
Centrifuge up to 4000×g for 1 min at − 15 °C in a Hettich Rotanta 460R centrifuge (or similar) with a swing bucket rotor for plates to settle plant material to the base of the wells.
Pause point
If the plates are not going to be processed at this time they can be stored at − 80 °C until processing is resumed.
Critical step
Care must be taken not to allow the samples to thaw prior to adding the homogenisation buffer as this has been shown to adversely affect DNA quality, particularly for recalcitrant species such as white clover.
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4.
Place the plates in an ice water bath for 20 min to warm to approximately 0 °C. Float them in a tin-foil receptacle to prevent ice adhering to the bottom of the plate.
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5.
Centrifuge at 4000×g for 1 min at − 15 °C to settle any distributed powdered plant material to the base of the wells. Carefully remove the seal and add 500 µl of pre-heated (55 °C for 10 min) HB buffer plus 1.8 µl of 20 µg µl−1 Proteinase K per well from a reservoir using a multichannel pipette. Dry the top surface of the plate and apply a fresh Thermal Bond heat seal. Mix well by shaking up and down manually (do not use a vortex machine), then centrifuge at 4000×g for 10 min at room temperature.
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6.
Transfer 300 µl of the supernatant into a fresh Axygen® 1.1 ml 96-well plate. Either automate with a liquid handling robot such as the Hamilton Microlab® STARlet or pipette manually.
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7.
Add 300 µl PB buffer. Apply an Axygen® sealing mat and mix the plates well by inverting and shaking manually for 10-20 s.
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8.
Incubate the plates in ice water bath for 15 min.
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9.
Centrifuge at maximum speed for 30 min at room temperature. For a Hettich Rotanta 460R centrifuge, maximum speed is 8595×g.
Pause point
The plates may be left at Step 9 for up to 72 h at 4 °C with no adverse effects on DNA quality.
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10.
Place a Pall AcroPrep™ Advance 96-Well 1 ml Filter plate on an Axygen® 1.1 ml 96-well deep well plate and add 600 µl BB buffer, followed by 400 µl supernatant per well. Mix well by pipetting up and down gently 10 times.
Critical step
Care is needed to avoid any possible cross-contamination due to overflow as each well is close to maximum volume. Additionally, gentle pipette mixing is required to prevent gDNA shearing.
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11.
Centrifuge for 2 min at 4000×g at room temperature. Discard the flow-through and dry the collection plate surface with a paper towel.
Critical step
With some plant species and tissues, such as ryegrass pseudostem, we have observed some clogging of filter membranes, likely due to carbohydrates. This has been reduced by removing an incubation step after addition of HB buffer. In the event that wells are clogged after centrifugation, this can be remedied by gently scraping or tapping the filter membrane with individual pipette tips followed by a repeat centrifugation.
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12.
Wash with 300 µl per well of BB buffer. Centrifuge at 4000×g for 2 min at room temperature.
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13.
Wash with 300 µl per well of WB buffer. Centrifuge at 4000×g for 2 min at room temperature.
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14.
Wash with 300 µl per well of absolute ethanol. Centrifuge at 4000×g for 2 min at room temperature. Discard flow-through.
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15.
Centrifuge at 4000×g for 5 min at room temperature to dry the membrane.
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16.
Swap to a fresh Axygen® 1.1 ml 96-well deep well plate for collection of the DNA to be eluted from the filter plate.
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17.
Add 115 µl of 10 mM Tris HCl pH 8 and 0.04 µl 100 mg ml−1 RNAse A per well (dispense from a reservoir) and centrifuge at 4000×g for 1 min at room temperature. This should yield approximately 100 µl of eluent containing 1–13 µg DNA per well, depending upon the plant species and grinding efficiency. Seal the plates for storage with a 4titude PCR sealing film.
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18.
The DNA is quantified with a Qubit® 2.0 Fluorometer, and 230–280 nm absorbance scans performed on a Nanodrop ND-1000 Spectrophotometer.
Timing
For one operator extracting two 96-well plates (192 samples):
Step 1, 1 h 40 min (loading tissue may be done in advance and stored for a week at 4 °C).
Steps 2–4, 30 min.
Step 5, 20 min.
Steps 6 + 7, 30 min.
Step 8, 15 min.
Steps 9 + 10, 1 h.
Steps 11–16, 30 min.
Freeze-dried/silica gel-dried tissue protocol
This protocol follows the steps described in the Fresh Tissue gDNA Extraction Protocol with the following changes.
Critical step
We have found that when freeze-drying, for best long-term plant tissue archiving and DNA quality, we place recently harvested material in a pre-chilled freeze-drier and apply an effective vacuum as soon as possible. This avoids a pre-freezing stage and the potential for tissue to thaw and cell components to rupture in the initial freeze-drying process, which we have found yields degraded gDNA. If freeze-drying facilities are not readily available, particularly if harvesting material in more remote field conditions, an effective alternative is drying plant tissue samples with silica gel. After 36 h of drying using a ratio of 10 g silica gel (LabServ Pronalys 2–6 mm self-indicating Silica gel) per 1 g fresh weight of plant material, sequencing-quality gDNA can be extracted using our modified protocol. This drying method is suitable for leaf material in small batches or 96-well plates. For both methods, fresh material may be stored in sealed bags for up to a week at 4 °C, rather than liquid nitrogen or dry-ice, prior to drying, with no reduction in DNA quality. Harvesting directly into the 96-well plate, drying, adding stainless steel balls, then sealing and storing in a container with silica gel to reduce humidity, provides an effective way to stockpile sample plates in advance of DNA extraction. Drying samples in the plates also avoids potential cross contamination if transferring previously dried, friable and likely electrostatically charged samples into wells.
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1.
Harvest 50 mg of fresh leaf directly into each well of a Corning® 1 ml deep well 96-well plate, and freeze-dry or silica gel-dry the entire 96-well plate and contents. If the material has already been dried, add 15 mg of dried tissue to each well. Two smaller (3/32 inch; 2.38 mm) stainless balls per well are used for the homogenisation step as the plant tissue is already brittle and readily ground. Heat seal with a Thermal Bond seal.
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2.
The next steps are identical to the Fresh tissue protocol except that the Qiagen TissueLyser adapters do not need to be pre-cooled, centrifugations may be performed at room temperature, and Steps 4 (ice bath) and 5 are omitted.
Individual tube tissue protocol
This protocol follows the steps described in the Fresh Tissue gDNA Extraction Protocol with the following changes.
Critical step
This protocol is effective with fresh and freeze/silica gel-dried plant tissue. It is important to match the bead size with the size/strength of the screw-cap microtube to prevent breakage. For cost-effectiveness and consistent gDNA quality, we use a Pall AcroPrep™ Advance 96-Well 1 ml Filter plate rather than individual silica filter spin columns. We use the same plate repeatedly for multiple individual extractions but use a fresh well for each extraction.
This protocol can be performed using option A (Fresh Tissue), or option B (Freeze/silica gel-dried Tissue).
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(A)
Using fresh plant tissue fresh material.
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1.
Harvest 50 mg of fresh leaf directly into a 2 ml Sarstedt Screw Cap microtube, add two 3.97 mm stainless steel balls, and float the tube in liquid nitrogen for 5 min. Similarly, pre-chill Qiagen TissueLyser tube adapters for microfuge tubes.
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2.
Place the tube in a Qiagen TissueLyser with tube adapter and grind tissue at 30 Hz for 1 min. Repeat if grinding is insufficient.
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3.
Centrifuge at maximum speed in an Eppendorf® Microcentrifuge 5415D benchtop centrifuge (or similar) with a fixed angle rotor to settle powdered plant material to the base of the tube. The protocol follows the same steps as the Fresh Tissue protocol from step 4 except that at step 6 the supernatant is transferred to a 1.5 ml Eppendorf microfuge tube, and a benchtop microcentrifuge is used until step 10.
Critical step
We found that using individual silica spin columns did not produce the required gDNA quality. Consequently, we use a Pall AcroPrep™ Advance 96-Well 1 ml Filter plate. For cost effectiveness, we use the same plate repeatedly for multiple individual extractions but use a fresh well for each extraction.
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(B)
Using freeze/silica gel-dried tissue.
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1.
Either harvest 50 mg of fresh leaf directly into a 2 ml Sarstedt Screw Cap microtube and freeze/silica gel dry as described above, or add 15 mg of previously dried tissue. Add a ¼ inch Ceramic Sphere.
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2.
Place the tube in a Qiagen TissueLyser with tube adapter and grind tissue at 30 Hz for 1 min. Repeat if grinding is insufficient.
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3.
Centrifuge at maximum speed in an Eppendorf® Microcentrifuge 5415D benchtop centrifuge (or similar) with a fixed angle rotor to settle powdered plant material to the base of the tube. The protocol follows the same steps as the Fresh Tissue protocol from step 6 except that at step 6 the supernatant is transferred to a 1.5 ml Eppendorf microfuge tube, and a benchtop microcentrifuge is used until step 10.
Critical step
We found that using individual silica spin columns did not produce the required gDNA quality. Consequently, we use a Pall AcroPrep™ Advance 96-Well 1 ml Filter plate. For cost effectiveness, we use the same plate repeatedly for multiple consecutive individual extractions but use a fresh well for each extraction.