Extraction of DNA from potato and phytobiome using CTAB protocols

Potato (Solanum tuberosum) is considered the fifth most important staple crop worldwide. Extracting DNA from important crops such as potatoes is challenging due to the presence of compounds like polysaccharides, polyphenols, and other secondary metabolites. These compounds, which accumulate in plants under stressful conditions, can interfere with DNA extraction and genetic analysis. The ability to isolate high-quality DNA, which includes nucleic acids from the host, pathogens, and microbiome, is essential for pathogen detection and molecular genetic studies to address food security and climate change. However, depending upon the objective, the approach to DNA extraction can differ.

Introducing desirable traits such as stress tolerance into potato plants enhances their resilience to stressors, leading to more stable and productive crop yields. With genetic studies, pinpointing traits often requires long read sequencing that depends on high molecular weight DNA. A research group (Morgan et al. 2023) developing transgenic lines overexpressing Escherichia coli OtsB gene to enhance their stress tolerance of potatoes utilized a cryogenic CTAB isolation method for DNA extraction for Southern blotting.

A major application of HMW DNA is for generating draft genomes. As an example, root-lesion nematodes (Pratylenchus) are economically important plant-parasitic nematodes infecting potatoes, though despite their significance, genome information related to the Pratylenchus genus is scarce. A team of researchers (Arora et al., 2023) reported the draft genome assembly of Pratylenchus scribneri, generated using the PacBio Sequel IIe System and ultra-low DNA input HiFi sequencing. To extract high molecular weight DNA, the nematode pellet was grounded using liquid nitrogen and subsequently dissolved in CTAB buffer (OPS Diagnostics, Lebanon, NJ,).

In comparison to methods for HMW DNA isolation, extracting DNA from plants and pathogens may require more efficient grinding. For instance, Ralstonia solanacearum causes bacterial wilt of potatoes, which is a major limiting factor in potato production as well as affecting other plants such as blueberries, may not be effectively ruptured using a mortar and pestle. A team of scientists (Bocsanczy et al. 2023) found that the best method to assess the feasibility of a simple, cost-effective, safe, and rapid diagnostic system with MiFi was the Synergy™ 2.0 Plant Extraction Kit (OPS Diagnostics, Lebanon, NJ) in comparison with Dneasy Plant Mini® Kit (Qiagen, Hilden) and a traditional CTAB protocol. However, the CTAB protocol followed the LN2 mortar and pestle protocol with CTAB extraction buffer (OPS Diagnostics catalog CEB 125-01) which enabled the highest DNA yield from woody plants such as blueberry. This finding led to the development of MiFi blueberry e-probes that effectively identified R. solanacearum strains pathogenic to blueberries with high specificity.

Efficient DNA extraction is also necessary for environmental DNA samples. The most important potato disease worldwide is late blight caused by Phytophthora infestans, a water mold (oomycete) that was the culprit behind the Irish Potato Famine of 1845 to 1852. A comprehensive metabarcoding study was conducted by a team of researchers (Redekar et al. 2023) to determine spatial and temporal dynamics of oomycete communities (Phytophthora, Pythium and Phytopythium) present in irrigation water collected from various sources within a commercial container nursery in Oregon over the course of one year. DNA from leaf disks of Rhododendron catawbiense 'Grandiflorum' plants were used as oomycete bait, and subsequently extracted using the Synergy Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ).

Below are two protocols that yield very different DNA preparations. Cryogenic grinding using liquid nitrogen and a CTAB extraction method yields high molecular weight DNA. A rapid protocol, in this case using the SYNERGY 2.0 Plant DNA Extraction Kit, is useful for isolating smaller DNA fragments in less time for diagnostics purposes.

Cryogenic Grinding Procedure for High Molecular Weight DNA for Long Read Sequencing

1. Use liquid nitrogen to chill and pulverize leaf tissue with a mortar and pestle. Weigh frozen powdered tissue (100 mg) into microfuge tubes.

2. Add 500 µl OPS Diagnostics CTAB Extraction Buffer to each tube.

3. Incubate the sample 60°C for 30 minutes.

4. Centrifuge the sample at 10,000 x g for 5 minutes. Transfer the supernatant to a new tube.

5. Add 5 µl RNase A solution to the supernatant and incubate at room temperature for 15 minutes.

6. Add an equal volume of chloroform/isoamyl alcohol to the solution, mix by vortexing, and then centrifuge for 1 minute at 13,000 x g. Transfer the upper aqueous phase to a clean microcentrifuge tube.

7. Repeat step 6 until the upper aqueous layer is clear.

8. Add 0.7 volume of isopropanol and incubate at -20°C for 15 minutes.

9. Centrifuge the tubes at 13,000 x g for 5 minutes to pellet DNA.

10. Carefully decant the liquid and wash the pellet with ice cold 70% ethanol. This step is done twice.

11. Place the pellet into the SpeedVac with the caps open. Run the SpeedVac on medium heat for about 90 seconds.

12. Resuspend the dry pellet in 100 µl of TE.

Rapid Synergy Procedure for Illumina Sequencing and PCR

This protocol uses the SYNERGY 2.0 Plant DNA Extraction Kit.

1. Add up to 50 mg of plant tissue (approximately 5 leaf punches) and 500 µl of Plant Homogenization Buffer to the 2 ml Homogenization Tubes.

2. Place the Homogenization Tubes into the bead beater and homogenize the sample at the highest speed for 1 minute. (If the plant sample is not completely homogenized, repeat the process, as the sample can occasionally press against the tube wall and avoid homogenization.When adequately processed, the tube will lack foam.)

3. Centrifuge the Homogenization Tubes at 15,000 x g for 5 minutes to pellet the debris, grinding matrix and contaminants. NOTE: The grinding matrix has an adsorption capacity for contaminants. For optimal results, the amount of starting plant tissue for some species should be reduced if the supernatant is not clear after centrifugation.

4. Transfer the clear supernatant into a clean Microfuge Tube.

5. To obtain RNA-free DNA, add 5 µl of RNase A Solution to the supernatant. Vortex for 5 seconds. Incubate at 37°C for 15 minutes.

6. Add 7/10 volume of isopropanol. Vortex for 5 seconds. Incubate at -20°C for 15 minutes.

7. Centrifuge the tube at 10,000 x g for 5 minutes to pellet the DNA. Decant the supernatant without disrupting the pellet.

8. Wash the pellet with 500 µl of ice cold 70% ethanol. Decant the liquid, then wash the pellet again with 500 µl of ice cold 70% ethanol. Decant the liquid and remove residual liquid.

9. Dissolve the DNA in Molecular Biology Grade water or TE buffer (if storing) and use for PCR or other applications. NOTE: Pellets can be difficult to resuspend.

10. Store at -80°C if storage of samples is required.


Arora, D., Hernandez, A. G., Walden, K. K. O., Fields, C. J., and Yan, G. 2023. First draft genome assembly of root-lesion nematode Pratylenchus scribneri generated using long-read sequencing. International Journal of Molecular Sciences. 24:7311

Bocsanczy, A. M., Espíndola, A. S., Cardwell, K. F., and Norman, D. J. 2023. Development and validation of e-probes with the mifi system for detection of Ralstonia solanacearum species complex in blueberries. PhytoFrontiers. 3:137-147

Morgan, B. L., Kakeshpour, T., Occhialini, A., King, G., Sichterman, M., Harbison, S. A., Rigoulot, S. B., Brabazon, H., Lenaghan, S. C., and Stewart, S. C. 2023. Heterologous expression of Otsb increases tuber yield and phenotypic stability in potato under both abiotic and biotic stresses. Plants. 12:3394

OPS-Diagnostics. CTAB Protocol for Isolating DNA from Plant Tissues; OPS-Diagnostics: Lebanon, NJ, USA, 2019.

Redekar, N., Eberhart, J. L., and Parke, J. L. 2019. Diversity of Phytophthora, Pythium, and Phytopythium species in recycled irrigation water in a container nursery. Phytobiomes Journal. 3:31-45

Solo, N., Kud, J., Dandurand, L., Caplan, A., Kuhl, J. C., and Xiao, F. 2021. Characterization of superoxide dismutase from the potato cyst nematode, Globodera pallida. Phytopathology. 111:2110-2117