参见作者原研究论文

本实验方案简略版
Jan 2019

本文章节


 

Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions
适于研究树-真菌互作的安全DNA提取方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host material (Picea abies, Populus trichocarpa, Pseudotsuga menziesii) infected with different pathogens (Heterobasidion annosum, Heterobasidion parviporum, Leptographium wagenerii, Sphaerulina musiva). Additionally, DNA was extracted from pure cultures of the pathogens and several endophytic fungi. PCR success rate was 100% for young poplar material and fungal samples, and 48-72% for conifer and mature broadleaved plant samples. We recommend using 10-50 mg of fresh sample for the best results. The method offers a safe and low-cost DNA extraction alternative to study tree-fungus interactions, and is a potential resource for teaching purposes.

Keywords: DNA extraction (DNA 提取), Plant DNA (植物DNA), Fungal DNA (真菌DNA), Forest pathology (森林病理学), Plant-microbe interactions (植物-微生物互作), Low-cost (低成本), Non-toxic (无毒害)

Background

DNA extraction is a central technique in plant-microbe interaction research and for plant disease diagnostic purposes. The abundance of plant-fungal interactions is reflected by the high numbers of cultivable strains isolated from plant samples (Arnold et al., 2001; Higgins et al., 2007; Terhonen et al., 2011). Processing of large numbers of samples for routine PCR reactions with commercial kits can be costly. Additionally, the requirement for hazardous chemicals, such as 2-mercaptoethanol, chloroform, or phenol can restrict the suitability of the protocols for teaching and training purposes.

Due to high secondary metabolite content, many plant samples, and in particular tree tissues, can pose challenges for nucleic acid extraction. Current protocols for DNA extraction from recalcitrant plant tissue utilize organic solvents (chloroform, 2-mercaptoethanol) or surfactants (e.g., cetyl trimethylammonium bromide) (Porebski et al., 1997; Chiong et al., 2017; Yi et al., 2018). Despite their benefits for DNA extraction, these chemicals pose hazards to user health and the environment. Several versions of low-cost, fast, and low health risk protocols for DNA extraction exist for mycelium (Chi et al., 2009), juvenile plant tissue (Edwards et al., 1991; Lu, 2011), grains (Saini et al., 1999), and dried plant tissues (Chabi Sika et al., 2015). However, these methods have not been applied to study tree-fungus interactions, and many times they have been tested only on limited number of sample types. Our goal was to develop and test a safe and low-cost DNA-extraction protocol that is suitable for extracting DNA from various tree tissues and tree-associated fungal samples. After sample preparation, the extraction takes about 60 min. The extracted DNA is suitable for PCR-based downstream applications, such as DNA-based pathogen detection with species-specific primers. Due to its safety and affordability, the method is also a potential resource for teaching and training purposes.

Materials and Reagents

  1. Standard materials and reagents
    1. Sterile microcentrifuge tubes, 1.5 or 2.0 ml
    2. Micropipette tips: 10, 200, 1,000 µl
    3. Miracloth (Calbiochem, e.g., VWR, catalog number: 475855-1 )
    4. Plant or fungal samples
    5. Purified (RO, DI, MilliQ, Nanopure) and sterilized water
    6. 100 bp DNA ladder (Jena Bioscience, catalog number: M-214S )
    7. 1 kb DNA ladder (New England Biolabs, catalog number: N0552 )
    8. NaCl
    9. KCl
    10. EDTA
    11. SDS
    12. Tris (pH 7.5)
    13. Polyvinypyrrolidone (PVP, CAS 900-39-8, FW 40,000, e.g., Caisson Labs, catalog number: P071-100GM )
    14. Isopropanol
    15. Ethanol (EtOH)
    16. Extraction buffer (see Recipes)
    17. Wash buffer (see Recipes)

  2. Special materials and reagents for different tissue homogenization optionsB. Special materials and reagents for different tissue homogenization options
    1. Option 1 for soft leaf tissue and mycelium: No special materials or reagents
    2. Option 2 for various plant tissue and mycelium, larger than 100 mg: Liquid nitrogen
    3. Option 3 for various plant tissue and mycelium, smaller than 100 mg: Bead beater tubes (e.g., Lysing Matrix I, MP Biomedicals, catalog number: 116918050-CF )
      Note: To reduce plastic waste and save on costs, the bead beater tubes can be washed and re-used (see Notes).

Equipment

  1. Standard equipment
    1. Scalpels
    2. Tweezers
    3. Spatulas
    4. Scale (e.g., Metler-Toledo, model: ML54T )
    5. Micropipettes: 1, 10, 100, 1,000 µl
    6. Heat block (e.g., VWR, catalog number: 12621-096 )
    7. Vortex (e.g., VWR, catalog number: 10153-838 )
    8. Microcentrifuge (e.g., Eppendorf, model: 5424 )
    9. One of the following to heat up water: Microwave, waterbath (e.g., VWR, model: WB05 ), or hot plate (e.g., VWR, catalog number: NO97042-642 )
    10. Freezer, -20 °C or -80 °C

  2. Special equipment for different tissue homogenization options
    1. Option 1 for soft leaf tissue and mycelium: No special equipment
    2. Option 2 for various plant tissue and mycelium, more than 100 mg:
      Dewar for liquid nitrogen
      Ceramic mortars and pestles
    3. Option 3 for various plant tissue and mycelium, less than 100 mg:
      Bead beater (e.g., Biospec, model: Mini-Beadbeater 16 , catalog number: 607/607EUR )
    4. For sampling xylem tissue from mature trees: chisel (e.g., Grainger, catalog number: 2AJA6 ) and mallet (e.g., Grainger, catalog number: 4YR61 ), cutting board

Procedure

  1. Sample preparation: Weigh 10-50 mg of sample (see Figure 1).
    1. Plant tissue: Use tweezers and scalpel to cut the sample to approximately 5 × 5 × 1 mm pieces. Smaller and thinner pieces will result in better sample quality.
    2. Mycelium: Use scalpel/spatula to scrape mycelium from the surface of Petri plates, or use spatula to collect mycelium from liquid culture.
    3. Xylem tissue from mature trees: Place wood sample on cutting board. Use chisel and mallet to harvest pieces of xylem tissue. Cut to 5 × 5 × 1 mm pieces with scalpel and spatula.
    Note: For mycelium, minimize the amount of agar for higher DNA quality.


    Figure 1. Examples of sample sizes for DNA extraction. A. Fresh phloem from 8-week-old Populus trichocarpa trees. Weight 20 mg. B. Fresh phloem from mature P. trichocarpa trees. Weight 15 mg. C. Fresh Leptographium wagnerii mycelium harvested from liquid malt extract cultures. Weight 20 mg. Interval between vertical lines = 1 mm.

  2. Homogenize sample and add extraction buffer
    Three options are available depending on sample type. Complete homogenization is not necessary.
    1. Option 1: Mycelium and soft leaf tissue
      1. Place sample in a 1.5 or 2.0 ml microcentrifuge tube.
      2. Add 1 ml extraction buffer.
      3. Vortex rigorously for 20 s.
      4. Proceed to Procedure C.
    2. Option 2: Various plant tissue and mycelium ≥ 50 mg:
      1. Grind sample in mortar with pestle and liquid nitrogen.
      2. Transfer 10-50 mg of homogenized sample with a spatula or by decanting to a 1.5/2.0 ml microcentrifuge tube.
      3. Add 1 ml extraction buffer.
      4. Vortex rigorously for 20 s.
      5. Proceed to Procedure C.
    3. Option 3: Various plant tissue and mycelium ≤ 50 mg:
      1. Transfer the sample into a 2.0 ml beat beater tube.
      2. Add 1 ml extraction buffer.
      3. Process for 20 s in a bead beater.
      4. Proceed to Procedure C.
      Note: Use 1 ml of extraction buffer per 50 mg or less tissue. To allow sufficient vortexing, use only 1 ml of buffer per tube.

  3. DNA extraction
    1. Heat the wash buffer in a water bath or in a beaker with warm water (approximately 65 °C) to dissolve any precipitants. Temperature is not critical, as long as no precipitants remain. Mix by inversion.
    2. Lysis and debris elimination: Incubate the samples in extraction buffer at 65 °C for 15 min. Vortex once during incubation. Centrifuge at 6,000 x g for 10 min.
    3. Eliminate debris: After centrifugation, transfer ca. 0.5 volume of the supernatant into a new tube. Add 1 volume of pre-heated wash buffer, and vortex samples for 20 s. Centrifuge at 21,000 x g for 10 min.
      Note: Complete debris elimination is not critical when pipetting the supernatant.
    4. Precipitate: Transfer ca. 0.7 volume of supernatant into a new tube, add 0.85 volume of isopropanol (room temperature), and mix by inversion for 20 s. Centrifuge at 21,000 x g for 10 min.
      Note: Minimize transferring any debris while pipetting the supernatant.
    5. Wash the pellet: Pour out the supernatant, and remove remaining supernatant by tapping the tubes upside down on a paper towel. Add 200 µl of 70% ethanol, and centrifuge at 21,000 x g for 5 min.
      Note: Consider local regulations for correct handling of isopropanol waste.
    6. Dry the pellet: Pipet out the ethanol. Leave the caps open, and dry pellets in a heat block at 65 °C for 5 min.
      Note: For faster drying, remove as much of the ethanol as possible.
    7. Resuspension: Dissolve the pellet in 20-50 µl of TE buffer or nuclease free water. Vortex to dissolve if needed, and centrifuge briefly to collect any droplets to the bottom of the tube. Store DNA samples at -20 °C or -80 °C until used.
      Note: If the DNA pellet is not colorless or white post 70% ethanol wash, add 20-50 µl of TE buffer or nuclease-free water to resuspend the DNA without disturbing the pellet. Gently pipet the liquid a few times in the tube and collect the supernatant as DNA for downstream processes. Keep the pellet until DNA is quantified. Use the DNA for PCR-based detection, or store in freezer until used.

Data analysis

Analysis of protocol performance:

  1. Plant and fungal material used for DNA extractions
    To test the suitability of the protocol, we extracted DNA from artificially inoculated trees, naturally infected trees, and mycelium (Table 1). The artificially inoculated samples included 8-week-old Populus trichocarpa plants spray-inoculated with Sphaerulina musiva (LeBoldus et al., 2010; Abraham et al., 2018), and 3-year-old Picea abies plants plug-inoculated with Heterobasidion sp. (Terhonen et al., 2019). The naturally infected plant samples included stem cankers on mature P. trichocarpa trees caused by S. musiva infection, and mature Pseudotsuga menziesii roots infected with Leptographium wageneri. Necrotic or discolored phloem or xylem samples with 10-380 mg of tissue (average 86 mg) were used for DNA extraction. For every sample, 1 ml of extraction buffer was used regardless of sample weight. Fungal DNA was extracted from mycelium harvested from pure cultures (Table 1). For solid medium, either malt extract agar (2% malt extract, 2% agar) or KV8 agar (18% V8 juice, 0.2% CaCO3, 2% agar) were used. Cultures on solid medium were grown in ambient room temperature. For liquid medium, either malt extract medium (2% malt extract) or KV8 medium (18% V8 juice, 0.2% CaCO3) was used (Table 1). Cultures in liquid medium were grown in ambient room temperature on a rotary shaker (100-150 rpm). The mycelium was harvested from liquid medium by filtering through Miracloth (Calbiochem) and rinsed with DI water.

  2. Assessment of DNA sample quality
    DNA concentrations were measured with Nanodrop, Nanophotometer, or Qubit. PCR, quantitative real-time PCR (qPCR), agarose gel electrophoresis (Figure 2), and fungal ITS sequencing were used to evaluate sample quality. For PCR and qPCR, no-template negative controls and positive template controls were included into each run to evaluate detection reliability.
      For the P. trichocarpa samples that were inoculated or naturally infected with S. musiva, we used a host-pathogen specific assay (Abraham et al., 2018). For detection of Heterobasidion species from inoculated wood samples, species-specific primers for H. annosum and H. parviporum (Hantula and Vainio, 2003) were used (Terhonen et al., 2019). For detection of L. wageneri from P. pseudotsuga roots and fungal cultures, we used Leptographium-specific primers (Schweigkofler et al., 2005). Primers for P. menziesii (Winton et al., 2002) were used to distinguish PCR-inhibition from negative samples, and to amplify DNA extracted from Douglas-fir needles.
      DNA samples from Diplodia sapinea cultures were amplified with primers targeting the nuclear large subunit, elongation factor and calmodulin regions (Vilgalys and Hester, 1990; Carbone and Kohn, 1999; Grünig et al., 2007; Nelsen et al., 2011). Additionally, DNA from D. sapinea and fungal endophytes was amplified with primers ITS1-F and ITS4 for the fungal ribosomal internal transcribed spacer region (White et al., 1990; Gardes and Bruns, 1993). The PCR conditions are specified in Table 2 and primer sequences in Table 3. All PCR amplicons were visualized under UV light on 1.5% agarose gels with StainINTM RED or GelRedTM nucleic acid stains. For fungal species used for sequencing, the PCR products were purified and sequenced using the respective primers (Table 1) at Microsynth SEQLAB (Göttingen, Germany).
      The effect of potential PCR inhibitors in the DNA samples on target detection was evaluated with a multiplex Taqman qPCR protocol (Abraham et al., 2018). DNA extracted with a commercial kit (DNeasy Plant Mini, Qiagen) from comparable tissue samples was used as a reference for low-inhibitor samples. Seven-point dilution series were prepared for P. trichocarpa (10-fold dilution series, 60-6 × 10-4 ng/µl) and S. musiva DNA samples (5-fold dilution series, 50-3.2 × 10-3 ng/µl) (Abraham et al., 2018) extracted with the developed method and with the commercial kit. The quantification cycle (Cq) values for the samples from the two extraction methods were compared, to evaluate the impact of potential PCR inhibitors on target detection.


    Figure 2. Examples of DNA samples extracted with the developed protocol. A. Three Diplodia sapinea DNA samples extracted from mycelium grown on malt extract agar (MEA). Lanes 1, 3, and 5: 50-500 ng of DNA. Lanes 2, 4, and 6: 10-fold dilutions of samples in lanes 1, 3, and 5. Gel: 2.0% agarose in 1× TAE, 100 V, 35 min. Ladder: 100 bp DNA ladder. B. DNA extracted from Leptographium wagnerii mycelium from liquid malt extract (lane 1), Sphaerulina musiva mycelium grown on KV8 agar (lane 2), S. musiva grown in liquid KV8 medium (3), fresh poplar phloem (lane 4), fresh poplar leaves (lane 5), mature Douglas-fir xylem (lane 6), and fresh Douglas-fir needles (lane 7). Lanes 1-4 and 6-7: 20-100 ng DNA. Lane 5: 500 ng DNA. Gel: 1% agarose in 1× TAE, 120 V, 70 min. Ladder: 1 kb DNA ladder.

    Table 1. Sample types extracted with the protocol, PCR success rates (%), and number of sequenced samples
    aGardes and Bruns 1993, White et al., 1990
    bGrünig et al., 2007
    cCarbone and Kohn, 1999
    dVilgalys and Hester, 1990, Nelsen et al., 2011

    Table 2. PCR conditions and primers used to test sample quality

    aCarbone and Kohn 1999, Grünig et al., 2007
    bCarbone and Kohn, 1999
    cVilgalys and Hester, 1990, Nelsen et al., 2011
    dGardes and Brunns, 1993; White et al., 1990
    eHantula and Vainio, 2003
    fSchweigkofler et al., 2005
    gAbraham et al., 2018
    hWinton et al., 2002

    Table 3. Primer sequences used in the PCR reactions.

    aCarbone and Kohn, 1999, Grünig et al., 2007
    bCarbone and Kohn, 1999
    cVilgalys and Hester, 1990, Nelsen et al., 2011
    dGardes and Brunns, 1993, White et al., 1990
    eHantula and Vainio, 2003
    fSchweigkofler et al., 2005
    gAbraham et al., 2018
    hWinton et al., 2002

  3. Data analysis
    Data analysis was conducted in R version 3.6.1. The effect of PCR inhibitors on quantification cycle (Cq) values was estimated using ANOVA followed by Tukey’s HSD tests (Figure 3A). We visualized the contribution of sample weight, DNA concentration, and sample purity (A260/280 and A260/230) on PCR success by plotting the results from principal component analysis (PCA) for the plant and fungal samples (Figures 3B-3D). The PCA results were visualized with the R package factoextra (Kassambara and Mundt 2017). For P. abies samples, necrosis length was also included in the model. Separate PCA’s were computed for P. trichocarpa samples (n = 85), P. abies samples (n = 48), and fungal samples (n = 129). Differences in template properties between failed and successful PCR reactions within the same sample type were compared by two-sample t-tests. If necessary, data were normalized with log-transformations.

  4. Protocol performance
    All the DNA samples extracted from 8-week-old P. trichocarpa phloem inoculated with S. musiva were PCR-positive for the pathogen (Table 1). PCR amplification worked for all fungal DNA samples, and the PCR products were suitable for fungal ITS sequencing. In comparison, PCR success rate was lower for DNA samples extracted from naturally infected mature P. trichocarpa samples (72%), inoculated 3-year-old P. abies samples (48%), and naturally infected mature P. menziesii roots (60%) (Table 1). The extraction protocol yields total DNA with the majority of the fragments larger than 10 kb (Figure 2). The samples are stable at least for 2 years in -20 °C. Partial DNA fragmentation (Figure 2B) did not affect PCR performance.
      Based on the comparison of Cq values for DNA samples extracted with a commercial kit, the extracted DNA samples may contain inhibitors that can affect the accuracy of target quantification by qPCR (Figure 3A). The Cq values were lower for the three highest dilutions (P < 0.040). After 1,000-fold dilution, the extraction method had no effect on quantification. The standard curves prepared from the DNA samples extracted with the protocol had lower amplification efficiencies compared to the commercial kit (Figure 1A). It is possible that the qPCR protocol in Abraham et al. (2018) is not optimal for the samples extracted with the developed DNA extraction protocol. However, the extraction method did not affect target detection.


    Figure 3. Comparative qPCR analysis to estimate the presence of sample inhibitors (A), and principal component analysis to visualize sample properties in Populus trichocarpa samples (B), young Picea abies samples (C), and fungal samples (D)

      We explored the association of DNA sample properties with PCR success by visualizing the results from principal component analysis (PCA). The two first principal components (PC) explained 68-87% of the total variation. The A260/280 and A260/230 values explained majority of variation along PC1 for P. trichocarpa, P. abies, and fungal DNA samples (55%, 85% and 75% contribution to PC1 variation, respectively) (Figures 1B-1D). For P. trichocarpa, the samples split into two groups on both sides of the vertical PC2 axis (Figure 3B). For the samples on the left side of the PC2 axis, PCR success rate was 100% and sample purity was relatively high (Figure 3B).
      Sample weight explained 55% and 40% of the variation along the PC2 in P. trichocarpa and P. abies, respectively. Larger initial sample weights were associated with lower PCR success rate for mature poplar and young Norway spruce DNA samples (Table 4). Among the 34 mature poplar samples that clustered on the right side of PC2 axis (Figure 3B), initial sample weights and DNA concentrations were higher for the failed PCR reactions (t = 2.882, P = 0.007 and t = 2.195, P = 0.035, respectively). Similarly, initial sample weights and DNA concentrations were higher for P. abies samples that failed PCR amplification (t = 1.9511, P = 0.057 and t = 2.060, P = 0.045, respectively). For the mature poplar samples, PCR failure both for host and pathogen amplification despite higher DNA concentration is probably associated with low template quality combined with high polyphenolic content in the mature bark tissue samples. For Norway spruce, PCR failure for pathogen amplification despite high DNA concentration and sufficient template quality is probably explained by low amount of pathogen DNA in less colonized samples. Alternatively, high phenolic content in heavily colonized samples may have inhibited pathogen detection, as increasing necrosis length was associated with lower PCR success in Norway spruce samples. This indicates that DNA samples extracted from tree tissue samples with high amounts of lignin or other polyphenolic compounds may have lower PCR success rates.
      The fungal DNA samples were highly variable based on A260/230 and A260/280 values (75% of variation on PC1, Figure 3D). The DNA samples extracted from mycelium grown in liquid cultures typically had higher measures of purity compared to fungal samples with agar (Table 4). Based on this, we recommend minimizing the amount of agar in the fungal samples used for DNA extractions. Despite high variation in sample purity, PCR amplification was successful for all the fungal DNA samples.

    Table 4. Sample weight, DNA concentrations, and DNA absorbance values for different DNA sample types extracted with the protocol. Median, minimum and maximum values are indicated.

    *Both host and pathogen PCR failed
    **Only pathogen PCR tested

Notes

  1. Amount of extraction buffer and sample weight
    We recommended to use 1 ml of extraction buffer per 50 mg or less of plant or fungal tissue. Low buffer volume relative to extracted tissue can have a negative effect on PCR success.
  2. Dissolving the pellet
    If the DNA pellet is not colorless or white post 70% ethanol wash, add 20-50 μl of TE buffer or nuclease-free water to resuspend the DNA without disturbing the pellet. Gently pipet the liquid a few times in the tube and collect the supernatant as DNA for downstream processes. Keep the pellet until DNA is quantified. Use the DNA for PCR-based detection, or store in freezer until used.
  3. Re-using the bead beater tubes
    To reduce costs and plastic waste, the bead beater tubes can be washed, treated with bleach to degrade DNA, autoclaved, and re-used. Separate the beads, caps and tubes in separate containers. Fill the containers with warm soap water, agitate for 5 min, and pour out the soap water. Rinse with tap water until runoff is clear. Shake tubes and caps to remove remaining tap water. Rinse twice with DI-water. To remove any remaining DNA, soak the components 1 h in 3% w/v NaOCl solution (1:1 solution with commercial bleach and DI-water) (Kemp and Smith 2005). Rinse twice with tap water, followed by two DI-water rinses. Let the tube components dry overnight, or dry in an oven. Once the components are dry, compile the tubes and autoclave at 121 °C for 30 min.

Recipes

  1. Extraction buffer
    1 M NaCl
    100 mM Tris HCl
    10 mM EDTA
    2% PVP
    1. Mix all ingredients in a beaker on a stirring hot plate
    2. Fill to desired volume with sterile purified water
    3. Heat the solution until PVP is dissolved
  2. Wash buffer
    1% SDS
    0.5 M KCl or NaCl
    1. Mix all ingredients in a beaker on a stirring hot plate
    2. Fill to desired volume with sterile purified water
    3. Heat the solution until no precipitation is visible
    4. Heat the wash buffer before use to dissolve any precipitants

Acknowledgments

This research was supported by the DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0018196, and by funding from the Faculty of Forest Sciences and Forest Ecology, University of Göttingen. Jumoke Aduke Babalola and David Robert Rӑscuţoi, University of Göttingen, are highly acknowledged for their contribution to the DNA extraction and PCR experimental set up. Dr. Patrick Bennett is thanked for providing the L. wagenerii strains and naturally infected Douglas-fir root samples, and Dr. Kelsey Søndreli for providing the mature P. trichocarpa samples naturally infected with S. musiva.

Competing interests

No competing interests declared.

References

  1. Abraham, N. D., Chitrampal, P., Keriö, S. and LeBoldus, J. M. (2018). Multiplex qPCR for detection and quantification of Sphaerulina musiva in Populus stems. Plant Pathol 67(9): 1874-1882.
  2. Arnold, A. E., Maynard, Z. and Gilbert, G. S. (2001). Fungal endophytes in dicotyledonous neotropical trees: patterns of abundance and diversity. Mycol Res 105(12): 1502-1507.
  3. Carbone, I. and Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91(3): 553-556.
  4. Chabi Sika, K., Kefela, T., Adoukonou-Sagbadja, H., Ahoton, L., Saidou, A., Baba-Moussa, L., Jno Baptiste, L., Kotconi, S. O. and Gachomo, E. W. (2015). A simple and efficient genomic DNA extraction protocol for large scale genetic analyses of plant biological systems. Plant Gene 1: 43-45.
  5. Chi, M. H., Park, S. Y. and Lee, Y. H. (2009). A quick and safe method for fungal DNA extraction. Plant Pathol J 25: 108-111. 
  6. Chiong, K. T., Damaj, M. B., Padilla, C. S., Avila, C. A., Pant, S. R., Mandadi, K. K., Ramos, N. R., Carvalho, D. V. and Mirkov, T. E. (2017). Reproducible genomic DNA preparation from diverse crop species for molecular genetic applications. Plant Methods 13(1): 106.
  7. Edwards, K., Johnstone, C. and Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19(6): 1349. 
  8. Gardes, M. and Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts. Mol Ecol 2(2): 113-118. 
  9. Grünig, C. R., Brunner, P. C., Duo, A. and Sieber, T. N. (2007). Suitability of methods for species recognition in the Phialocephala fortinii-Acephala applanata species complex using DNA analysis. Fungal Genet Biol 44(8): 773-788. 
  10. Hantula, J. and Vainio, E. (2003). Specific primers for the differentiation of Heterobasidion annosum (s.str.) and H. parviporum infected stumps in northern Europe. Silva Fennica 37. 
  11. Higgins, K. L., Arnold, A. E., Miadlikowska, J., Sarvate, S. D. and Lutzoni, F. (2007). Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages. Mol Phylogenet Evol 42(2): 543-555. 
  12. Kassambara, A., and Mundt, F. (2017). factoextra: Extract and visualize the results of multivariate data analyses.
  13. Kemp, B. M. and Smith, D. G. (2005). Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Sci Int 154(1): 53-61. 
  14. LeBoldus, J. M., Blenis, P. V. and Thomas, B. R. (2010). A method to induce stem cankers by inoculating nonwounded Populus clones with Septoria musiva spore suspensions. Plant Dis 94(10): 1238-1242. 
  15. Lu, Y. (2011). Extract genomic DNA from Arabidopsis leaves (can be used for other tissues as well). Bio-protocol 1(13): e90.
  16. Nelsen, M. P., Lücking, R., Mbatchou, J. S., Andrew, C. J., Spielmann, A. A. and Lumbsch, H. T. (2011). New insights into relationships of lichen-forming Dothideomycetes. Fungal Diversity 51(1): 155-162.
  17. Porebski, S., Bailey, L. G. and Baum, B. R. (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15(1): 8-15.
  18. Saini, H. S., Shepherd, M. and Henry, R. J. (1999). Microwave extraction of total genomic DNA from barley grains for use in PCR. J Inst Brew 105(3): 185-190.
  19. Schweigkofler, W., W.J, O., S.L, S., D.R, C., Maeda, K., Peay, K. and Garbelotto, M. (2005). Detection and quantification of Leptographium wageneri, the cause of black-stain root disease, from bark beetles (Coleoptera: Scolytidae) in Northern California using regular and real-time PCR. Canadian Journal of Forest Research 35: 1798-1808. 
  20. Terhonen, E., Langer, G. J., Bußkamp, J., Rӑscuţoi, D. R. and Blumenstein, K. (2019). Low water availability increases necrosis in Picea abies after artificial inoculation with fungal root rot pathogens Heterobasidion parviporum and Heterobasidion annosum. Forests 10(1): 55.
  21. Terhonen, E., Marco, T., Sun, H., Jalkanen, R., Kasanen, R., Vuorinen, M. and Asiegbu, F. (2011). The effect of latitude, season and needle-age on the mycota of Scots pine (Pinus sylvestris) in Finland. Silva Fenn 45
  22. Vilgalys, R. and Hester, M. (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172(8): 4238-4246.
  23. White, T., Bruns, T., Lee, S., Taylor, J., Innis, M., Gelfand, D. and Sninsky, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols, Academic Press, Inc., pp. 315-322.
  24. Winton, L. M., Stone, J. K., Watrud, L. S. and Hansen, E. M. (2002). Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction. Phytopathology 92(1): 112-116.
  25. Yi, S., Jin, W., Yuan, Y. and Fang, Y. (2018). An optimized CTAB method for genomic DNA extraction from freshly-picked pinnae of fern, Adiantum capillus-veneris L. Bio-protocol 8(13): e2906.

简介

[摘要 ] 我们提出了一种安全,低成本的方法,适用于从菌丝体和树木组织样品中提取DNA。样品制备后,提取过程大约需要60分钟。通过从各种树木组织样品和菌丝体中提取DNA来测试方法性能DNA从少年提取和成熟主体材料(云杉冷杉,胡杨毛果,黄杉花旗松感染不同病原体()Heterobasidion annosum ,Heterobasidion parviporum ,Leptographium wagenerii ,Sphaerulina musiva )。额外的,DNA从提取纯的病原体和几种内生真菌培养物。年轻杨树材料和真菌样品的PCR成功率是100%,针叶树和成熟阔叶植物样品的PCR成功率是48-72%。我们建议使用10-50 mg的新鲜样品最好结果。该方法为研究树-真菌相互作用提供了一种安全且低成本的DNA提取方法,并且是潜在的提纯方法。兴的目的。

[背景 ] DNA提取是植物-微生物相互作用研究和植物疾病诊断purposes.The丰植物真菌相互作用的中央技术由高数量从植物样品中分离的培养的菌株的反射(阿诺德等人,2001;希金斯等人,2007; Terhonen 等人,2011)。使用商业试剂盒处理大量样品进行常规PCR反应的成本可能很高,此外,对2-巯基乙醇,氯仿或苯酚等有害化学品的需求也可能增加。限制协议在教学和培训中的适用性。

由于高的二次代谢物含量,许多植物样品,并且在特定的树组织中,可以构成为从顽抗植物组织提取DNA核酸extraction.Current协议挑战利用有机溶剂(氯仿,2-巯基乙醇)或表面活性剂(Ë 。(例如,十六烷基三甲基溴化铵)(Porebski 等,1997;Chiong 等,2017; Yi 等,2018)。尽管这些化学品对DNA提取有好处,但对使用者的健康和环境构成危害。低成本,快速,以及用于DNA提取存在菌丝低健康风险协议(驰。等人,2009) ,幼年型植物组织(爱德华兹等人,1991;路,2011) ,谷物(赛尼。等人, (1999年)和干燥的植物组织(Chabi Sika 等人,2015年)。然而,这些方法尚未用于研究树-真菌相互作用,许多时候仅对有限数量的样品类型进行了测试。开发和测试一种安全且低成本的DNA提取试剂盒 适用于从各种树木组织和与树木相关的真菌样品中提取DNA的rotocol。样品制备后,提取需要大约60分钟的时间。提取的DNA适用于基于PCR的下游应用,例如基于DNA的病原体检测特异性引物:由于其安全性和可承受性,该方法也是用于教学和培训目的的潜在资源。

关键字:DNA 提取, 植物DNA, 真菌DNA, 森林病理学, 植物-微生物互作, 低成本, 无毒害

材料和试剂


 


标准材料和试剂
1. 1.5或2.0 ml无菌微量离心管      


2. 微量移液器吸头:10、200、1,000 µl      


3. Miracloth (Calbiochem ,例如,VWR,目录号:475585-1)      


4. 植物或真菌样品      


5. 纯化(RO,DI,M IlliQ ,Nanopure )和消毒水      


6. 100 Bp DNA阶梯(Jena Bioscience,目录号:M-214S)      


7. 1 Kb DNA梯子(新英格兰生物实验室,目录号:N0552)      


8. 氯化钠      


9. 氯化钾      


10. EDTA   


11. 安全数据表   


12. Tris(pH 7.5)   


13. 聚乙烯吡咯烷酮(PVP,CAS 900-39-8,FW 40,000,ē 。G. ,沉箱实验室,目录号:P071-100GM)   


14. 异丙醇   


15. 乙醇(EtOH)   


16. 提取缓冲液(请参见配方)   


17. 洗涤缓冲液(请参阅配方)   


 


用于不同组织均质化选择的特殊材料和试剂
叶片软组织和菌丝体的选择1:无特殊材料或试剂
各种植物组织和菌丝体的选择2,大于100 mg:液氮
3对于各种选项植物组织和菌丝体,小于100的Mg:珠打浆机管(ê 。G. ,裂解矩阵I,MP Biomedicals公司,目录号:116918050-CF )
注意:为减少塑料浪费并节省成本,可对珠子搅拌器管进行清洗和重新使用(请参阅“注释” )。






设备


 


标准装备
手术刀
镊子
铲子
秤(例如,Metler -Toledo,型号:ML54T)
微量移液器:1,10,100,1,000 µl
加热块(例如,VWR,目录号:12621-096)
涡流(例如,VWR,目录号:10153-838)
微量离心机(例如,Eppendorf,型号:5424)
加热水的下列方法之一:微波,水浴(例如,VWR,型号:WB05)或加热板(例如,VWR,目录号:NO97042-642)
冷冻室,-20 °C或-80 °C
 


用于不同组织均质化选择的专用设备
叶片软组织和菌丝体的选择1:无特殊设备
对于各种植物组织和菌丝体,大于100毫克的选项2:
杜瓦瓶液氮


陶瓷研钵和杵


对于各种植物组织和菌丝体,小于100毫克的选项3 :
打珠机(例如, Biospec ,型号:迷你型珠磨器16,目录号:607 / 607EUR)


采样木质部从成熟的树木:钻探(ē 。G. ,固安捷,目录号:2AJA6)和马利特(ē 。G. ,固安捷,目录号:4YR61),切菜板
 


程序


 


样品制备:称量10-50 mg样品(见图1)。
植物组织:用镊子和手术刀将样品切成约5×5×1 mm的块,较小和较薄的块将使样品质量更好。
菌丝体:使用手术刀/ 刮刀从P Etri板表面刮除菌丝体,或使用刮刀从液体培养物中收集菌丝体。
成熟树木的木质部组织:将木材样品放在砧板上,用凿子和木槌收获木质部组织碎片,用手术刀和刮刀切成5×5×1 mm的碎片。
注意:对于菌丝体,请尽量减少琼脂的用量以提高DNA质量。


 


 


1.实施例图样本规模的DNA 提取。甲。新鲜韧皮部从8周龄胡杨毛果树。重量20的Mg。乙。新鲜韧皮部从成熟P. 毛果树。重量15的Mg。Ç 。新鲜Leptographium Wagnerii 菌丝体从液态麦芽提取物中收获,重量20毫克,垂直线之间的间隔= 1毫米。


 


均质样品并添加提取缓冲液
根据样品类型,可以使用三个选项,无需完全均质化。


选项1:菌丝体和软叶组织
将样品放入1.5或2.0 ml微量离心管中。
加入1毫升提取缓冲液。
剧烈涡旋20 s。
继续执行程序C。
选项2:各种植物组织和菌丝体≥50 mg:
用研钵和液氮在研钵中研磨样品。
用刮刀或倾析到1.5 / 2.0 ml微量离心管中,转移10-50 mg均质样品。
加入1毫升提取缓冲液。
剧烈涡旋20 s。
继续执行程序C。
选项3:≤50 mg的各种植物组织和菌丝体:
将样品转移到2.0 ml打浆器管中。
加入1毫升提取缓冲液。
搅珠机处理20 s。
继续执行程序C。
注意:每50 mg或更少的组织中应使用1 ml提取缓冲液。为了充分涡旋,每管仅使用1 ml缓冲液。


 


DNA提取
在水浴或烧杯中用温水(约65 °C )加热洗涤缓冲液以溶解任何沉淀剂,温度并不严格,只要没有残留的沉淀剂即可。
消除裂解和碎片:将样品在提取缓冲液中于65 °C 孵育15 分钟。在孵育期间涡旋一次,以6,000 xg离心10分钟。
消除碎片:离心后,将约0.5 体积的酵母转移到新试管中,加入1体积的预热洗涤缓冲液,并将样品涡旋20秒钟,以21,000 xg离心10分钟。
注意:移去污渍时,彻底清除碎片不是关键。


沉淀:传输约在21000 0.7体积漫到一个新的管中,加入0.85体积的异丙醇(室温),并且通过反转20秒混合离心。XG 为10分钟。  
注意:在移取收集的液体时,应尽量减少转移任何碎屑。


洗涤沉淀物:倒出上清液,倒置试管,将其倒在纸巾上以除去残留的污染物。加入200 µl 70%乙醇,并以21,000 xg离心5分钟。
注意:请考虑当地法规,以正确处理异丙醇废物。


干燥沉淀物:移出乙醇,将瓶盖打开,将沉淀物在65 °C 的加热块中干燥5分钟。
注意:为了更快地干燥,请尽可能多地除去乙醇。


重悬液:将沉淀物溶于20-50 µl TE缓冲液或无核酸酶的水中。涡旋溶解(如果需要),短暂离心以收集液滴到试管底部。将DNA样品储存在-20 °C 或-80 ° C C 直到使用。
Ñ OTE:如果DNA沉淀不是无色或白色柱70%乙醇洗净,加入20-50 μ TE的升缓冲液或无核酸酶的水重悬DNA而不干扰pellet.Gently在吸管的液体数次试管收集上清液作为DNA用于下游过程,保持沉淀直至DNA定量。将DNA用于基于PCR的检测,或储存在冰箱中直至使用。


 


资料分析


 


协议性能分析:


用于DNA提取的植物和真菌材料
为了测试协议的适用性,我们提取的DNA从人工接种树木,自然感染树和.The人工接种样品包括8周龄菌丝(表1)杨毛果与植物喷雾接种Sphaerulina musiva (LeBoldus 等人。,2010;亚伯拉罕。等,2018) ,和3年之久的云杉冷杉植物插件接种Heterobasidion SP。(Terhonen 等,2019),包括茎溃疡成熟的自然感染植物样品。P. 毛引起树木由S. musiva 感染,和成熟的黄杉花旗松感染根Leptographium wageneri .Necrotic 或变色韧皮部或木质部样品用10-380 mg组织(平均86毫克)用于DNA extraction.For每个样品,将1ml的无论样品重量如何,均使用提取缓冲液。从纯培养物收获的菌丝体中提取真菌DNA(表1)。对于固体培养基,可以使用麦芽提取物琼脂(2%麦芽提取物,2%琼脂)或KV8琼脂(18%V8汁) ,0.2%使用CaCO 3 (2%琼脂),在室温下在固体培养基上培养,对于液体培养基,分别使用麦芽提取物培养基(2%麦芽提取物)或KV8培养基(18%V8汁液,0.2%CaCO 3 )。在室温下,在旋转摇床(100-150 rpm)上培养液体培养基中的培养物,并通过Miracloth (Calbiochem )过滤从液体培养基中收获菌丝体,并用去离子水冲洗。


 


DNA样品质量评估
使用Nanodrop,Nanophotometer或Qubit 测量DNA浓度.PCR,定量实时PCR(qPCR),琼脂糖凝胶电泳(图2)和真菌ITS测序用于评估样品质量。每次运行均包含模板阴性对照和阳性模板对照,以评估检测的可靠性。


  为P. 毛果其是在样品oculated或自然感染S. musiva ,我们使用了宿主-病原体特异性测定(亚伯拉罕等人,2018)。对于检测Heterobasidion 从接种的木材小号物种amples,物种特异性引物为H. annosum 和H. parviporum (Hantula 和Vainio ,2003)被用于(Terhonen 等人,2019) 。对于检测L. wageneri 从P. 黄杉根和真菌培养物中,我们使用Leptographium 特异性引物(Schweigkofler 等等人,2005)。孟席斯疟原虫的引物(Winton 等人,2002)被用于区分阴性样品中的PCR抑制作用,以及扩增从花旗松针中提取的DNA。  


用针对核大亚基,延伸因子和钙调蛋白区域的引物扩增  来自双翅目培养物的DNA样品(Vilgalys和Hester,1990;Carbone和Konn ,1999;Grünig 等,2007; Nelsen 等,2011)。用真菌核糖体内部转录间隔区的引物ITS1-F和ITS4扩增出D. sapinea 和真菌内生菌的DNA (White et al。,1990; Gardes and Bruns ,1993).PCR条件列于表2和表3中的引物序列。所有PCR扩增子均在带有StainIN TM RED或GelRed TM 核酸染料的1.5%琼脂糖凝胶上的紫外光下可视化。对于用于测序的真菌物种,PCR产物使用相应的引物进行纯化和测序(表1)在Microsynth SEQ LAB(德国哥廷根)。


  使用多重Taqman qPCR方案(Abraham 等,2018)评估了DNA样品中潜在的PCR抑制剂对靶标检测的影响。使用商业试剂盒(DNeasy Plant Mini,Qiagen)从可比的组织样品中提取的DNA 作为用于低抑制剂样品七点稀释系列的引用是对用于制备P. 毛(10倍稀释系列,60 - 6 × 10 -4 纳克/微升)和S. Musiva DNA样品(5倍稀释系列, 50-3.2 × 10 -3 ng /μl (Abraham et al。,2018)用开发的方法和商业试剂盒进行提取。比较两种提取方法的样品的定量循环(Cq )值,以评估潜在的PCR抑制剂对靶标检测的影响。


 


 


图2.与开发中提取的DNA的样品的例子。编protocol.A 三孢梢。从菌丝体生长在麦芽提取物琼脂(MEA)中提取的DNA的样品泳道1,3和5:50-500 ng的DNA的泳道2。 ,4,和6:样品的10倍稀释液在泳道1,3和5凝胶:在1×TAE,100琼脂糖2.0%。V,35分钟爬梯:100bp的DNA梯状条带.B 。DNA提取自Leptographium wagnerii 从液体麦芽提取物(泳道1),菌丝Sphaerulina musiva 菌丝上生长KV8 琼脂(泳道2),S. musiva 在液体生长KV8 介质(3),新鲜杨树韧皮部(泳道4),新鲜叶杨(泳道5) ,成熟的道格拉斯冷杉木质部(第6道)和新鲜的道格拉斯冷杉针(第7道)。泳道1-4和6-7:20-100 ng DNA。泳道5:500 ng DNA。凝胶:1%琼脂糖1×TAE,120 V,70分钟梯子:1 kb DNA阶梯。


 


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                                                                 


表1.用方案提取的样品类型,PCR成功率(%)和测序样品的数量


样品类型


DNA样品中的真菌


DNA样品中的宿主


样品描述


样本年龄


测试样品


PCR成功率(%)


排序样本


从植物材料中提取的DNA


异源异化


青海云杉


接种韧皮部


3年


20


70


未排序


细小异花孢


青海云杉


接种韧皮部


3年


20


二十五


Leptographium wageneri


黄杉花旗松


从根部自然感染的木质部


几年





60


Sphaerulina musiva


毛果杨


接种干叶


8周


9


78


接种韧皮部


8周


48


100


自然感染韧皮部


几年


34


72


未感染


毛果杨


树叶


1周


四个


100


黄杉花旗松


针头


1年


6


100


从菌丝体中提取的DNA


针叶树根内生菌


不适用


麦芽汁琼脂菌丝体


2周


二十四


100


ITS1 一个:14个样品ITS4 一个:10个样品


洋蛇


不适用


麦芽汁琼脂菌丝体


2周


39


100


ITS1 一个:17个样品ITS4 一个:22个样品


3周


二十二


100


22Pf_EF1α_ - [R b


4个星期


二十二


100


22个,带CAL-737R c


5周


二十二


100


22带LR6 d


Leptographium wageneri


不适用


菌丝,液体麦芽提取液


3周





100


未排序


榆粳稻内生菌


不适用


麦芽汁琼脂菌丝体


2周


61


100


ITS4 a :61个样本


Sphaerulina musiva


不适用


带有KV8琼脂的菌丝体


1周


9


100


未排序


 


菌丝体,液体ID KV8 培养基


1周


二十五


100


未排序


a Gardes and Bruns 1993,White 等人,1990             


b 格鲁尼格等人,2007年


c Carbone 和Kohn ,1999年             


d Vilgalys 和Hester ,1990年,Nelsen 等,2011年             






 


表2.用于测试样品质量的PCR条件和引物


检测物种


底漆


热循环仪设置


反应体积,微升


反应组成


1)初始变性


2)退火和伸长


3)最终伸长率


蒂pinea


EF1-728F和Pf_EF1α_R 一


95°C 3分钟


30×95°C 30 s,49°C 1分钟,72°C 1分钟


72°C 10分钟


二十五


反应混合1:
1×PCR缓冲液
(含氯化钾或2 NH 4 ·SO 4 ),
1.5毫摩尔MgCl 2 ,
200倍μM的dNTP,
0.5μM的每种引物,
1.25U的innuTaq DNA聚合酶(Alytik 耶拿AG),100纳克模板DNA的






CAL-228F和CAL-737R b


30×95°C 30 s,50°C 1分钟,72°C 1分钟


nu-LSU-287-5'-mpn和LR 6 c


30×95°C 30 s,48°C 1分钟,72°C 1分钟


ITS1-F和ITS4 d


A)15×95°C 30 s,55°C 1分钟,72°C 1分钟
B)15×95°C 30 s,63°C 1分钟,72°C 1分钟


内生真菌


ITS1-F和ITS4 d


95°C 3分钟


A)15×95°C 30 s,55°C 1分钟,72°C 1分钟
B)15×95°C 30 s,63°C 1分钟,72°C 1分钟


72°C 7分钟


二十五


异源化


KJ-F和KJ-R或
MJ-F和MJ-R e


95°C 10分钟


40×95°C 30 s,67°C 35 s,72°C 1分钟


72°C 7分钟


二十五


Leptographium wagnerii


LEPTO1和LEPTO2 f


94°C 4分钟


35×94°C 15 s,65°C 25 s,72°C 40 s


72°C 10分钟


二十五


反应混合2:
1X标准的Taq
的MgCl 2 -free缓冲液,
1.5毫摩尔MgCl 2 ,
200倍μM的dNTP,
每种引物为0.2μM,
1.25U的Taq DNA聚合酶
(New England Biolabs公司),
15-100纳克DNA模板


毛果杨


eIF4F1和eIF4F1-R g


94°C 4分钟


35×94°C 15 s,58°C 20 s,72°C 30 s


72°C 10分钟


15


Sphaerulina musiva


NABtF 和NABtR g


9 4°C 4分钟


35×94°C 15 s,58°C 20 s,72°C 30 s


72°C 10分钟


15


黄杉花旗松


LFY989F和LFY1102R h


94°C 4分钟


35×94°C 15 s,55°C 30 s,72°C 40 s


72°C 10分钟


二十五


反应混合物2 ,但每个引物含
0.4 µM


a Carbone and Kohn 1999,Grüniget al。,2007             


b Carbone 和Kohn ,1999年                           


c Vilgalys and Hester ,1990年,Nelsen 等,2011年             


d Gardes 和Brunns ,1993 ;White 等,1990。             


Ë Hantula 和Vainio ,2003             


f Schweigkofler 等,2005             


g Abraham 等人,2018             


h Winton 等,2002






 


表3. PCR反应中使用的引物序列。


引物对


DNA模板的起源


正向引物序列(5'-> 3')


反向引物序列(5'-> 3')


EF1-728F和Pf_EF1α_R 一


蒂pinea


CATCGAGAAGTTCGAGAAGG


GGGTTGTAGCCAACCTTCTTG


CAL-228F和CAL-737R b


蒂pinea


GAGTTCAAGGAGGCCTTCTCCC


CATCTTTCTGGCCATCATGG


nu-LSU-287-5'-mpn和LR 6 c


蒂pinea


CGAGTTGTTTGGGAATGC


CGCCAGTTCTGCTTACC


ITS1和ITS4 d


菌类


CTTGGTCATTTAGAGGAAGTAA


TCCTCCGCTTATTGATATGC


KJ-F和KJ-R,或


细小异花孢


CCATTAACGGAACCGACGTG


GTGCGGCTCATTCTACGCTATC


MJ-F和MJ-R e


异源异化


GGTCCTGTCTGGCTTTGC


CTGAAGCACACCTTGCCA


LEPTO1和LEPTO2 f


ept 属


CAAAGACGGCAGACGCGAGTCTC


GTTCCAGGGAACTCGGAAG


eIF4F1和eIF4F1-R g


杨属。


TGGGGCCTCTATTTAGCATGGAT


CTGCACCCGAAATGGGATTGACC


NABtF 和NABtR g


Sphaerulina musiva


CGACCTGAACCACCTTGTCT


CACGGTAACAGCGCGGAACGA


LFY989F和LFY1102R h


黄杉花旗松


TGTTCAACATCCAGGCAATGA


TAACCGGCGCCTGAATGCTTCG


a Carbone and Kohn ,1999,Grünig 等,2007             


b Carbone 和Kohn ,1999年                           


c Vilgalys and Hester ,1990年,Nelsen 等,2011年             


d Gardes and Brunns ,1993,White 等,1990             


Ë Hantula 和Vainio ,2003             


f Schweigkofler 等,2005             


g Abraham 等人,2018             


h Winton 等,2002


 


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                


数据分析
在R版本3.6.1中进行了数据分析。使用ANOVA进行评估,然后使用Tukey的HSD测试(图3 A )估算PCR抑制剂对定量循环(C q )值的影响。我们可视化了样品重量,DNA浓度,通过绘制植物和真菌样品的主成分分析(PCA)的结果(图s 3 B - 3D )绘制PCR成功的样品纯度(A 260/280 和A 260/230 )。řPackage该Factoextra (Kassambara 而蒙特2017) 。对于P. 冷杉样品,坏死长度也包括在模型中。分离PCA'S计算了P. 毛果样品(N = 85),P. 冷杉样品(N = 48)以及真菌样本(n = 129)。通过两次样本t检验比较同一样本类型中失败和成功的PCR反应之间的模板性质差异,必要时使用对数转换对数据进行归一化。


 


协议性能
所有的8周龄提取的DNA样品P. 毛果韧皮部接种S. musiva 进行PCR阳性病原体(表1).PCR 扩增工作的所有真菌类DNA样品,并且将PCR产物适合于真菌ITS相比较而言,PCR成功率为用于从天然感染的成熟中提取的DNA的样品低P. 毛果样品(72%),接种3岁P. 冷杉样品(48%),和自然感染的成熟P. 花旗松根(60%)(表1)。提取方案产生的总DNA大部分片段大于10 kb(图2)。样品在-20 °C 至少稳定2年。部分DNA片段化(图2) 2 B )不影响PCR性能。 


  根据使用商业试剂盒提取的DNA样品的C q 值的比较,提取的DNA样品中可能含有可能影响通过qPCR进行目标定量的准确性的抑制剂(图3 A )。三种最高稀释度的Cq 值较低。 (P <0.040)。稀释1000倍后,提取方法对定量没有影响。从方案提取的DNA样品制备的标准曲线与市售试剂盒相比具有较低的扩增效率(图1A )。亚伯拉罕(Abraham)等人(2018)中的qPCR方案可能不适用于使用已开发的DNA提取方案提取的样品,但是该提取方法并未影响目标检测。


 


 


图3.比较qPCR分析来估算样品抑制剂的存在(A),和主成分分析,以可视化样本性质杨毛果样品(B),年轻云杉云杉SAMP LES(C),和真菌样品(d)。


 


  我们通过可视化主成分分析(PCA)的结果探索了DNA样品特性与PCR成功的关系。两个第一主成分(PC)解释了总变异的68-87%.A 260/280 和A 260 / 230个值一起为PC1说明大多数变异P. 毛果,P. 冷杉和真菌的DNA样品(55%,至85 PC1%和75%的贡献分别变化,)(图小号1个乙-1 d )。对于P. 毛果癣,样品在垂直PC2轴的两侧分为两组(图3 B )。对于PC2轴左侧的样品,PCR成功率为100%,样品纯度相对较高(图3 B))。


  样品重量解释55%和沿着在PC2的变化的40%的P. 毛果和P. 冷杉分别。较大的初始样品重量用下PCR成功率相关联,用于成熟白杨和年轻挪威云杉的DNA样品(表4)。在34个聚集在PC2轴右侧的成熟杨树样品中(图3 B ),失败的PCR反应的初始样品重量和DNA浓度较高(t = 2.882,P = 0.007和t = 2.195,P = 0.035类似地,PCR扩增失败的欧洲冷杉(P. abies)样品的初始样品重量和DNA浓度较高(分别为t = 1.9511,P = 0.057和t = 2.060,P = 0.045)。尽管DNA浓度较高,但宿主和病原体扩增均失败,这可能与模板质量低下以及成熟树皮组织样品中多酚含量高有关。集中度较低和模板质量足够高可能是由于定殖较少的样品中病原体DNA含量低所致。定殖严重的样品中酚类含量高可能抑制了病原体的检测,因为坏死长度的增加与挪威云杉样品中PCR成功率的降低有关。表明从树木组织样品中提取的含有大量木质素或其他多酚化合物的DNA样品可能具有较低的PCR成功率。


  真菌DNA样品基于A 260/230 和A 260/280 值(PC1变异的75%,图3 D )变化很大,与液体培养相比,从菌丝体中提取的DNA样品的纯度通常更高。基于此,我们建议尽量减少用于DNA提取的真菌样品中的琼脂含量。尽管样品纯度差异很大,但所有真菌DNA样品的PCR扩增都是成功的。


 


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                                                                


 


表4.根据协议提取的不同DNA样品类型的样品重量,DNA浓度和DNA吸光度值。显示了中位数,最小值和最大值。


样品类型


样品(重量,毫克)


 


DNA(ng / µl)


 


一个260/280


 


一个260/230


中位数


最小-最大


 


中位数


最小-最大


 


中位数


最小-最大


 


中位数


最小-最大


杨树,PCR可以


100


100


 


二十二


11-49


 


2.2


1.6-2.2


 


1.8


1.1-2.1


杨树成熟,PCR可以


20


10-160


 


3


1-15


 


1.6


0.9-2


 


0.5


0.2-1.2


杨树成熟,PCR失败*


35


15-140


 


7


1-20


 


1.3


1.2-2


 


0.4


0.3-0.6


云杉幼嫩,PCR OK


92


36-380


 


30


5-266


 


1.5


0.7-2


 


0.5


0.1-2


云杉年轻,PCR失败**


136


58-326


 


63


10-265


 


1.6


1.2-2.2


 


0.8


0.3-2.2


真菌,液体培养基


不适用


不适用


 


35


5-330


 


2.1


1.8-2.2


 


1.7


1.1-2.2


真菌,固体培养基


不适用


不适用


 


20


2-168


 


1.8


0.9-2.2


 


0.5


0.1-2.2


*宿主和病原体PCR均失败


**仅通过病原体PCR检测


 


版权所有©20 20 的作者;专用特许生物协议LLC 1                                                                                                                             




说明:logonew                


笔记


 


提取缓冲液量和样品重量
我们建议每50 mg或更少的植物或真菌组织使用1 ml提取缓冲液。相对于提取组织而言,缓冲液体积低可能会对PCR成功产生负面影响。


溶解沉淀
如果在70%乙醇洗涤后DNA沉淀不是无色或白色,则添加20-50 用微升TE缓冲液或无核酸酶的水重悬DNA,而不会干扰沉淀物。将液体轻轻移入试管中几次,并收集上清液作为DNA用于下游过程。保持沉淀物直至DNA定量。使用DNA用于基于PCR的检测,或保存在冰箱中直至使用。


重复使用搅拌器
为了降低成本和减少塑料浪费,可以清洗打珠机的管子,用漂白剂处理以降解DNA,高压灭菌后再使用,将珠子,瓶盖和管子分开放在单独的容器中,用温肥皂水填充容器,搅拌均匀。 5分钟,倒出肥皂水,用自来水冲洗直到清澈的水为止,摇动试管和盖子以除去残留的自来水,用去离子水冲洗两次,以除去任何残留的DNA,将成分浸泡3%1小时W / V NaOCl 溶液(使用商业漂白剂和去离子水的1:1溶液)(肯普和史密斯,2005年)。用自来水冲洗两次,然后进行两次去离子水冲洗,使试管组件隔夜干燥或在管中干燥。烘箱中。一旦组分是干的,编译管和高压釜在121 ℃下30分钟。                                         


 


菜谱


 


提取缓冲液
1 M氯化钠


100毫米Tris HCl


10毫米EDTA


2%PVP


在搅拌的热板上将所有成分混合在烧杯中
用无菌纯净水填充至所需体积
加热溶液直至PVP溶解
洗涤缓冲液
1%SDS


0.5 M 氯化钾或氯化钠


在搅拌的热板上将所有成分混合在烧杯中
用无菌纯净水填充至所需体积
加热溶液,直到看不到沉淀为止
使用前先加热洗涤缓冲液以溶解任何沉淀物
致谢


 


这项研究是由科学能源部办公室生物和环境研究(BER),授予no.DE-SC0018196办公室的支持,并通过森林科学的哥廷根。Jumoke大学学院和森林生态,资助Aduke 巴巴洛拉和大卫罗伯特Rӑscuţoi ,哥廷根大学,高度认可了他们的DNA提取贡献,PCR实验小号等了。帕特里克·班尼特博士感谢提供L. wagenerii 株和自然感染的道格拉斯冷杉根样品,和凯尔博士SOND RELI 提供成熟P. 毛样品自然感染S. musiva 。


 


利益争夺


 


没有宣布利益冲突。


 


参考文献


 


亚伯拉罕,ND,Chitrampal 。,P,安装Kerio 。,S和LeBoldus 。,JM(2018)多重qPCR用于检测和定量Sphaerulina musiva 在胡杨茎。植物病理学67(9):1874至1882年。
阿诺德,AE,梅纳德,Z.和吉尔伯特,GS(2001)。在双子叶植物新热带树木内生真菌:丰富性和多样性的格局 Mycol RES 105(12):1502至07年。
Carbone,I.和Kohn,LM(1999)。一种设计用于丝状子囊菌种形成研究的引物对的方法。Mycologia 91(3):553-556。
察必皇后西卡,K.,Kefela ,T。,Adoukonou-Sagbadja ,H。,Ahoton ,L。,赛义杜,A。,巴巴-穆萨,L.,JNO 巴普蒂斯特,L.,Kotconi ,SO和Gachomo ,EW(2015一种简单有效的基因组DNA提取方案,用于植物生物系统的大规模遗传分析(植物基因1:43-45)。
志,MH,公园,SY和Lee,Y. H.(2009)。快速和真菌DNA提取安全的方法。植物病理学Ĵ 25:108-111。              
Chiong ,KT,Damaj ,MB,Padilla,CS,Avila,CA,Pant,SR,Mandadi ,KK,Ramos,NR,Carvalho,DV和Mirkov ,TE(2017)。从多种作物物种中可再生的基因组DNA制备,用于分子遗传学的应用程序。植物的方法13(1):106。
Edwards,K.,Johnstone,C.和Thompson,C.(1991)。一种用于PCR分析的植物基因组DNA制备的简单快速方法。核酸研究19(6):1349。              
Gardes ,M。和布伦斯,TD(1993)。ITS引物具有增强的对担子菌的特异性-应用到菌根和锈的识别。分子ECOL 2(2):113-118。              
的Gr ü NIG,CR,布伦纳,PC,铎,A。和塞伯,TN(2007)。对于在物种识别方法适宜Phialocephala fortinii-羽衣applanata 物种复合使用DNA分析。真菌遗传学生物化学44(8):773 -788。              
Hantula ,J。和Vainio ,E。(2003)。特异性引物分化Heterobasidion Annosum (S.Str。)和H. Parviporum 感染树桩在北欧。 Silva的Fennica 37。              
希金斯,KL,阿诺德,AE,Miadlikowska ,J。,Sarvate ,SD和Lutzoni ,F。(2007).Phylogenetic 关系,主机亲和力,和寒带的地理结构和北极内生菌来自三个主要植物谱系。分子Phylogenet EVOL 42( 2):543-555。              
Kassambara ,A。和Mundt ,F。(2017).factoextra:提取并可视化多元数据分析的结果。
Kemp,BM和Smith,DG(2005)。使用漂白剂消除骨骼和牙齿表面的污染DNA。法医科学154(1):53-61。              
LeBoldus ,JM,Blenis ,PV和Thomas,BR(2010)。一种通过将未受伤的杨树克隆接种家蝇孢子悬浮液来诱导茎弯曲的方法,植物病 94(10):1238-1242。              
Lu,Y.(2011)。从拟南芥叶中提取基因组DNA (也可用于其他组织)。生物协议1(13):e90。
Nelsen,MP,Lucking ,R.,Mbatchou ,JS,Andrew,CJ,Spielmann ,AA和Lumbsch ,HT(2011)。形成地衣菌的关系的新见解。真菌多样性51(1):155-162。
Porebski ,S.,Bailey,LG和Baum,BR(1997)。对含有高多糖和多酚成分的植物的CTAB DNA提取方案的修改,植物分子生物学15(1):8-15。
Saini,HS,Shepherd,M.和Henry,RJ(1999)。微波从大麦粒中提取总基因组DNA,用于 PCR。J Inst Brew 105(3):185-190。
Schweigkofler ,W.,WJ,O.,SL,S.,DR,C.,Maeda,K.,Peay ,K。and Garbelotto ,M。(2005)。检测和定量化ept (Leptographium payneri),这是黑皮病的原因。定期和实时PCR ,从北加州的树皮甲虫(鞘翅目:鞘翅目)染色的根病。加拿大森林研究杂志35:1798-1808。              
E. Terhonen,E.,Langer,GJ,Busskamp,J.,DR S Scutoi,DR和Blumenstein,K.(2019)。真菌根腐病病原体人工接种小孢子菌和小核糖异菌后人工接种后,低水利用率增加了P ICEA abies的坏死。。 森林10(1):55。
E.Terhonen,T.Marco,Sun H.,Jalkanen,R.Kasanen,R.Vuorinen,M。和Asiegbu(2011)。纬度,季节和针头年龄对苏格兰松树(Sinus sylvestris )的真菌菌群,席尔瓦·芬恩45
Vilgalys ,R。和赫斯特,M。(1990)。从几个快速遗传鉴定和全球扩增核糖体DNA的映射隐球菌物种。 Ĵ 细菌学172(8):4238-4246。
White,T.,Bruns,T.,Lee,S.,Taylor,J.,Innis,M.,Gelfand,D.和Sninsky ,J.(1990)。真菌核糖体RNA基因的扩增和直接测序用于系统发育。在:PCR Protocols ,Academic Press,Inc。,第315-322页。
Winton,LM,Stone,JK,Watrud ,LS和Hansen,EM(2002)。用实时聚合酶链反应同时对宿主和病原体DNA进行单管定量。植物病理学92(1):112-116。
Yi,S.,Jin ,W。,Yuan,Y. and Fang,Y.(2018)。一种优化的CTAB方法,用于从新鲜采摘的蕨类植物印度铁线蕨(Adiantum capillus-veneris L.Bio -protocol)8中提取基因组DNA 。 13): e2906 ..
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用:Keriö, S., Terhonen, E. and LeBoldus, J. M. (2020). Safe DNA-extraction Protocol Suitable for Studying Tree-fungus Interactions. Bio-protocol 10(11): e3634. DOI: 10.21769/BioProtoc.3634.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。