The overall goal of this experiment is to compare two methods used to isolate high quality organellar DNA, both plastid and mitochondria DNA from plant leaf tissue that is suitable for next-generation sequencing. These methods can help answer key questions in plant biology such as the extent of organellar diversity across taxa and how changes in organellar genome sequence or structure affect development and stress response. We will compare two techniques, the differential centrifugation method that enriches for one specific organellar type and the meta fractionation technique that recovers total organellar DNA from smaller frozen tissue samples.
The standard procedure for growing wheat seedlings, is to plant seeds in vermiculite, in square pots with four to six seeds per corner. Transfer the pots to a greenhouse with a 16 hour light cycle. Water the plants each day.
Fertilize the plants with one quarter teaspoon of granular 20-20-20 MPK fertilizer upon germination, and again at seven days post germination. For adulation of wheat seedlings, plant seeds and care for plants as in the standard procedure. But place the pots in a dark growth chamber.
An alternative to growing these seedlings at a dark growth chamber is to cover the plants in the greenhouse but with proper ventilation. The first step in this procedure is to isolate the organelles. Harvest five grams of fresh tissue and rinse it in cold, sterile water in a chilled beaker on ice.
Using scissors, cut the leaf tissue into approximately one centimeter pieces directly into a pre-chilled 50 millimeter tube containing two ceramic grinding cylinders. Keep the samples on ice throughout the procedure. To avoid cross-contamination, it is important to change the scissors between samples.
Take the samples to the fume hood and add 20 milliliters of STE buffer to each 50 milliliter tube. Place the samples into pre-chilled cryogenic grinding blocks in a tissue grinding apparatus and grind the samples for 30 seconds at 1, 750 rotations per minute. Place the samples on ice for about one minute.
Rotate the sample positions and grind for another 30 seconds at 1, 750 rotations per minute. Return the samples to the ice bucket. For each sample, insert a funnel into a clean 50 milliliter tube placed in ice and place one layer of filtration cloth into the funnel.
Pre-wet the filtration cloth with five milliliters of STE buffer and save the flow through. Pour the homogenized tissue into the funnel. Rinse the grinding tube with 15 milliliters of STE buffer, recap and invert the tube to rinse walls and lid and pour its contents into the funnel.
Carefully remove the ceramic stones and then squeeze and wring out the filtration cloth into the funnel. To avoid cross-contamination, change gloves between samples. Wrap the tube caps with paraffin film to avoid spillage.
Centrifuge at 2, 000 x G, at four degree celsius for 10 minutes. Next, use a serological pipette to carefully aspirate the supernatant without disturbing the pellet and place it in a 50 milliliter high speed centrifuge tube with a tight sealing gasket. Place a small beaker of ice on a scale, tear the scale and weigh each supernatant.
Use STE buffer to balance the tubes to within 0.1 grams and then centrifuge at 18, 000 x G in four degree celsius for 20 minutes. When the centrifugation is complete, return samples to the fume hood and discard the supernatant. Add one milliliter of ST buffer to the pellet and resuspend gently using a soft paintbrush.
Add 24 milliliters of ST buffer to get a final volume of 25 milliliters. Gently swirl the paintbrush around in a suspension to mix, and then press the paintbrush on the side of a tube to remove all liquid before removing the paintbrush. After balancing the tubes, centrifuge at 18, 000 x G in four degree celsius for 20 minutes.
During this centrifugation, prepare DNA's 1 solution as described in the text protocol and aliquot 200 microliters to a 1.5 milliliter tube per sample. When the centrifugation is complete, discard the supernatant and blot the tube. To each high speed centrifuge tube, add 300 microliters of STE buffer and resuspend the pellet using a soft paint brush.
Place the paint brush in the previously prepared 1.5 milliliter tube containing 200 microliters of DNA's 1 solution and swirl the paint brush to remove any residual pellet stuck in the brush. Pipette the DNA's 1 solution into the high speed centrifuge tube and gently swirl to mix. Wrap paraffin film around the top of each tube and incubate at 37 degrees celsius in a water bath for 30 minutes.
During the incubation, gently mix by swirling. Using a pipette tip with a wide orifice, gently pipette the pellet mixture out of the tube and place it in a 1.5 milliliter low binding tube. Add 500 microliters of 400 millimolar EDTA, pH 8.0 to the high speed centrifuge tube.
Gently pipette to completely remove the residual pellet and transfer the EDTA to the low binding tube containing the pellet mixture. Gently mix by inversion. Centrifuge at 18, 000 x G, at four degree celsius for 20 minutes.
Subsequently, discard the supernatant, blot each tube and use the pellet immediately for DNA isolation. In this method, organellar DNA is enriched from total genomic DNA that was extracted using commercial DNA extraction columns. Prepare the required amount of MBD2 FC protein bound magnetic beads, per the manufacturer's instructions and scale the reactions to use between one and two micrograms of total input DNA.
In this demonstration, 160 microliters of beads is required for one microgram of total input DNA. To capture methylated nuclear DNA, add one microgram of input DNA to a tube containing 160 microliters of MBD2 bound magnetic beads for each individual sample and gently pipette up and down with a wide bore tip. Add the appropriate volume of 5x bind wash buffer for a final concentration of 1x and pipette the sample up and down with a wide bore tip, a few times to mix.
Rotate the tubes at room temperature for 15 minutes. For the success of the pull down, it is critical to thoroughly mix the beads during the incubation, particularly, if there is significant bead clumping. To prevent bead clumping and to ensure pull down of the methylated DNA, gently pipette the samples with a wide bore pipette tip and flick the samples two to three times throughout the incubation.
Briefly spin the tubes containing the DNA and magnetic bead mixture. Place the tubes on a magnetic rack for at least five minutes to collect the beads to the side of the tubes. The solution should appear clear, it contains un-methylated organellar enriched DNA.
Using wide bore tips, carefully remove the cleared supernatant without disturbing the beads and transfer the supernatant to a clean, low binding two milliliter microcentrifuge tube. Store the samples at 20 or 80 degrees celsius. Acute PCR assay was employed to assess the relative abundances of three organelle specific implicons in total genomic DNA and the organellar DNA fraction obtained from both methods.
Overall, the data indicate that the differential centrifugation method, preferentially enriches for mitochondria. The unmethylated fraction of the methylfractionated total genomic DNA, shows substantial enrichment of both organellar implicons. Percentages of reads mapped to the mitochondria or chloroplast chinese spring and chris cultivated wheat reference genomes are consistent with the QPCR results.
Differential centrifugation yield DNA that is more enriched in mitochondria DNA and methylfractionation yields DNA that likely reflects the native abundance of the two organellar genomes. Sample labels including E, designate idyllated samples and NE designate non-idyllated samples. Interestingly, idyllation did not change organellar abundances.
In both methods, the theoretical coverage of the mitochondria or chloroplast referenced genomes, exceeds 100x coverage, even when 12 libraries atremultiplexed. The total genomic DNA migrates as a diffused smear in post field gel electrophoresis and exceeds 50 kilobases. The nuclear fraction after methylfractionation decreases in size, but remains centered around 50 kilobases, suggesting that methylfractioanted DNA is most suitable for long read sequencing.
Once mastered, both organellar DNA extraction techniques can be done in one to two days, from sample collection to assessment of final product, if performed properly. The methylfractionation technique has the potential to greatly expand the range of organellar DNA studies to rare and difficult samples, since tissue inputs are extremely low. However, differential centrifugation may still be preferable when solely mitochondria DNA is required for downstream studies.
Likewise, centrifugation steps maybe adjusted to preferentially extract plastid DNA if desired. Both organellar DNA extraction methods yield DNA that is suitable for next generation sequencing but methylfractionation yields high molecular rate DNA that is ideal for long read sequencing.
