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Supplementary document 2. Method evaluation for DNA extraction and amplification from single fungal
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sporangia on freshwater phytoplankton
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Summary
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A strain of Zygorhizidium planktonicum (Chytridiomyocta) infecting the diatom Asterionella formosa was
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cultured to evaluate the PCR success rates in dependence of i) DNA extraction methods, ii) DNA polymerases,
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and iii) chytrid life cycle stages.
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Extraction of genomic DNA by hot alkaline or enzymatic lysis, and amplification with DNA polymerases KOD
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FX Neo or MightyAmp resulted in successful DNA amplification of a single chytrid sporangium in ~80% of all
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tested samples, although the success rate varied with life cycle stages.
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Experimental procedures
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Preparation of a chytrid strain and buffers
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A strain of Zygorhizidium planktonicum infecting the diatom Asterionella formosa (Fig. SD2.1) was isolated
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from the lower Columbia River, USA, in April 2011, and maintained by subsequent weekly transfers to
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uninfected host colonies (Kagami et al., 2010; Maier and Peterson, 2014). Before each experiment, each colony
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of A. formosa was isolated using a micropipette under an inverted microscope. Each cell was washed with
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sterile deionized water in an Utermöhl chamber, and subsequently transferred to a PCR tube with 2 µL of the
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sterile deionized water. Alkaline lysis buffer (25 mM NaOH and 0.2 mM of disodium EDTA, adjusted to pH
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12.0), neutralizing buffer (40 mM Tris-HCl, adjusted to pH 5.0), enzymatic lysis buffer (0.01% SDS, 0.1µg
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Proteinase K (Roche), 1 x Ex Taq PCR buffer (Takara)), and TE (10 mM Tris-HCl, 1mM EDTA, adjusted to pH
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8.0) were prepared locally.
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Experiment 1: Comparisons of DNA extraction methods (HS10 and HS30)
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The results of 2 procedures were compared: Exp1-HS10 and Exp1-HS30 (Table SD2.1). Thirty-two colonies of
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A. formosa with a single, intact sporangium of Z. planktonicum and 8 uninfected colonies of A. formosa
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(negative control) were collected (Fig. SD2.1). Five µL of alkaline lysis buffer were added to each sample tube.
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For Exp1-HS10, half samples (i.e., 16 infected samples and 4 uninfected samples) were incubated at 96˚C for 10
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min, here assigned as HS10 (Table SD2.2). For Exp1-HS30, the remaining samples were incubated at 96˚C for
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30 min (HS30; Table SD2.2). Additional 5 µL of neutralizing buffer were added to each sample tube. PCR was
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performed in a total volume of 6 µL consisting of 0.6 µL of DNA extract, 0.12 µL of KOD FX Neo DNA
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polymerase (1.0 U/µL; Toyobo), 3 µL of 2 x PCR Buffer for KOD FX Neo, 1.2 µL of 2 mM dNTPs, 0.12 µL of
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10 µM each primer of SI03 and SI16 (Supplementary document 1; Table S1), and 0.84 µL of
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sterile milliQ water, and in the thermal cycling conditions comprising an initial denaturation step (at 94˚C for 2
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min) and 45 cycles (at 98˚C for 10 sec, 53.5˚C for 30 sec, and 68˚C for 1min and 18 sec).
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Experiment 2: Comparisons of DNA extraction methods (HS10, EL, TS)
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The results of different DNA extraction methods (HS10, EL, and TS; Table SD2.2) were compared: Exp2-HS10,
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Exp2-EL, and Exp2-TS (Table SD2.1). Twenty-four colonies of A. formosa with a single, intact sporangium of
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Z. planktonicum and 6 uninfected colonies of A. formosa (negative control) were collected. Thereafter, one third
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of the samples (i.e., 8 infected samples and 2 uninfected samples) were subjected to the HS10 heating regimen
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(Table SD2.2) in 5 µL of alkaline lysis buffer, and thereafter neutralized by addition of 5µL of neutralizing
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buffer for Exp2-HS (Table SD2.1). The remaining samples were subjected to the EL heating regimen (Table
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SD2.2) in 10µL of enzymatic lysis buffer for Exp2-EL, and the TS heating regimen (Table SD2.2) in 10µL of
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TE buffer for Exp2-TS, respectively. PCR was performed for duplicate aliquots of each DNA extract in a total
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volume of 6 µL consisting of 0.6 µL of DNA extract, 0.12 µL of KOD FX Neo DNA polymerase (1.0 Unit/µL),
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3 µL of 2 x PCR Buffer for KOD FX Neo, 1.2 µL of 2 mM dNTPs, 0.12 µL of each primer (10 µM), i.e.,
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primers EF3 and EF4 (Smit et al., 1999), which were selected by in silico PCR analysis (Supplementary
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document 1; Table S1), and 0.84 µL of sterile milliQ water, and in the thermal cycling conditions comprising an
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initial denaturation step (at 94˚C for 2 min) and 35 cycles (at 98˚C for 10 sec, 48˚C for 30 sec, and 68˚C for 1
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min and 36 sec).
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Experiment 3: Comparisons of DNA polymerases
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In addition to the experiments using KOD FX Neo (Exp2-EL), aliquots of each EL extract (i.e., DNA extract
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used for Exp2-EL) were also amplified using other DNA polymerases: Exp3-MA an Exp3-ET (Table SD2.1).
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For each experiment, PCR was performed for duplicate aliquots of each EL extract according to the
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manufacturer’s protocol: for Exp3-MA, the PCR was performed in a total volume of 6 µL consisting of 0.6 µL
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of EL extract, 0.12 µL of MightyAmp DNA polymerase (1.25 U/µL; Takara), 3 µL of 2 x MightyAmp Buffer,
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0.18 µL of each primer (10 µM) EF3 and EF4, and 1.92 µL of sterile milliQ water, and in the thermal cycling
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conditions comprising an initial denaturation step (at 98˚C for 2min) and 35 cycles (at 98˚C for 10 sec, 60˚C for
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15 sec, and 68˚C for 1 min and 36 sec); for Exp3-ET, the PCR was performed in a total volume of 6 µL
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consisting of 0.6 µL of EL extract, 0.03 µL of Ex Taq HS (5 Unit/µL; Takara), 0.6 µL of 10 x Ex Taq Buffer,
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0.48 µL of 2.5 mM dNTPs, 0.3 µL of each primer (10 µM) EF3 and EF4, and 3.69 µL of sterile milliQ water,
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and in the thermal cycling conditions comprising 35 cycles (at 98˚C for 10 sec, 48˚C for 30 sec, and 68˚C for 1
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min and 36 sec).
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Experiment 4: Comparisons of life cycle stages
2
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In addition to the experiments using A. formosa with a single, intact sporangium of Z. planktonicum (Exp2-EL),
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DNA extract from 8 colonies for each of A. formosa with a single zoospore, 2 intact sporangia, and a single
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empty sporangium of Z. planktonicum (Fig. SD2.1) and from 6 uninfected colonies of A. formosa (negative
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control) were obtained by the EL heating regimen (Table SD2.2) in 10µL of enzymatic lysis buffer for Exp4-ZS,
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2IS, and ES (Table SD2.1). PCR was performed for duplicate aliquots of each DNA extract according to the
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same procedure used for Exp2-EL (i.e., PCR in a total volume of 6 µL including KOD FX Neo DNA
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polymerase and the primer set EF4/EF3 as described above).
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Post-PCR procedures
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After visualizing PCR products, excess primers and dNTPs were removed by using illustra ExoProStar (GE
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Healthcare) prior to commercial sequencing by FASMAC Co., Ltd. (Kanagawa, Japan). Statistical analysis was
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performed to compare the results of each experiment based on the generalized linear mixed model with a
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binomial error distribution (via the glmer function in the lme4 package version 1.1-7 for R) and Tukey’s
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multiple comparison tests (via the glht function in the multcomp package version 1.3-7 for R) setting the success
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or failure of amplification per each PCR sample as response variable, the difference of experimental procedures
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or life cycle stages as fixed effects of categorical explanatory variables, and the difference of DNA extracts used
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for PCR as random effects. The results of Exp3-ET were excluded from the analysis, because all PCR
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amplification attempts of Exp3-ET failed: the response variables consisting of only zero values in an
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experimental category cause errors during the analysis of the glmer function.
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Results
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No PCR product was amplified from the negative controls (i.e., the diatom A. formosa without chytrid infection)
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for any of the performed experiments. Numbers of the successfully analyzed samples were the same (13 out of
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16 samples) when performing two different experiments of the HotSHOT (HS) method (Exp1-HS10 and
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Exp1-HS30; Table SD2.1), which differed in heating time for the hot alkaline lysis (HS10 and HS30; Table
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SD2.2). Extending the heating time of HS above 10 minutes, however, did not improve the obtained outcome. In
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contrast, the fraction of successfully analyzed samples by using the thermal shock (TS) method was
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significantly lower than for the enzymatic lysis (EL) method (p = 0.00167) and the HS10 method (p = 0.00160)
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(Fig. SD2.2; Table SD2.3). The fraction of successfully amplified samples when using the DNA polymerases of
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KOD FX Neo (Toyobo) or MightyAmp (Takara) was quite similar (p=0.363; Table SD2.3), while no PCR
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product was obtained when using the Ex Taq HS DNA polymerase (Takara) (Fig. SD2.2). The fraction of
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successfully analyzed samples of a zoospore was significantly lower than of a single, intact sporangium (p =
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0.0181) and marginally lower than of two, intact sporangia (p = 0.0426) (Fig. SD2.2; Table SD2.3).
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Discussion
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Combining the HS10 or EL DNA extraction method with the KOD FX Neo or MightyAmp DNA polymerase
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for PCR amplification is efficient to analyze a single sporangium of Z. planktonicum (Fig. SD2.2). The TS
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extraction method was inefficient for fungal DNA extraction from a single colony of infected diatom, although
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TS was efficient for a single dinoflagellate cell (Kamikawa et al., 2007) and just heating (at 95˚C for 5min) was
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also enough for a single diatom cell (Lang and Kaczmarska, 2011). Consistently, Bärlocher et al. (2010) showed
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that freeze-thaw extraction is inefficient to analyze a single conidium of aquatic hyphomycetes: only one out of
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32 extractions was successfully sequenced. The difference in cell structure may be attributed to the difference in
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efficient method for DNA extraction between fungi and algae. HS10 (i.e., the HotSHOT method; Truett et al.,
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2000) has been applied to various organisms including zooplankton remains in lake sediments dated as more
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than 50 years ago (Ishida et al., 2012). EL is also used for various organisms including single pollen grains
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(Matsuki et al., 2007). According to manufactures’ instructions, the Ex Taq HS DNA polymerase is better in
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yield, sensitivity, and fidelity than a normal Taq polymerase. However, both of the KOD FX Neo and
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MightyAmp DNA polymerases have superior characteristics to facilitate amplifications of DNA from crude
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extracts containing PCR-inhibitory substances, such as unpurified DNA extracts of HS10, EL, and TS. The
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appropriate selection of specific PCR primers, DNA extraction methods, and DNA polymerases is crucial for
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genetic analysis of a single chytrid sporangium.
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Chytrid life cycle stages also affect the fractions of successfully analyzed samples (Fig. SD2.2). Z. planktonicum
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has two life cycle stages, i.e., a zoospore and a sporangium (Fig. SD2.1). After zoospores of Z. planktonicum are
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released from an individual sporangium, the empty sporangium is left on the cell wall of A. formosa. Then, a
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zoospore attaches to another A. formosa cell, and forms a new sporangium. Z. planktonicum on A. formosa may
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be at any life cycle stage of zoospore, intact sporangium, or empty sporangium. A zoospore is a single cell and
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smaller than a sporangium, which is an assemblage of multiple zoospores. Much lower quantity of fungal rDNA
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in a single zoospore than an intact sporangium may have caused lower success rates of their amplification.
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References
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Bärlocher, F., Charette, N., Letourneau, A., Nikolcheva, L.G., and Sridhar, K.R. (2010) Sequencing DNA
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extracted from single conidia of aquatic hyphomycetes. Fungal Ecology 3: 115–121.
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Ishida, S., Ohtsuki, H., Awano, T., Tsugeki, N.K., Makino, W., Suyama, Y., and Urabe, J. (2012) DNA
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extraction and amplification methods for ephippial cases of Daphnia resting eggs in lake sediments: a novel
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approach for reconstructing zooplankton population structure from the past. Limnology 13: 261–267.
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Kagami, M., Helmsing, N.R., and van Donk, E. (2010) Parasitic chytrids could promote copepod survival by
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mediating material transfer from inedible diatoms. Hydrobiologia 659: 49–54.
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Kamikawa, R., Hosoi-Tanabe, S., Yoshimatsu, S., Oyama, K., Masuda, I., and Sako, Y. (2007) Development of
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a novel molecular marker on the mitochondrial genome of a toxic dinoflagellate, Alexandrium spp., and its
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application in single-cell PCR. J Appl Phycol 20: 153–159.
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Lang, I., and Kaczmarska, I. (2011). A protocol for a single-cell PCR of diatoms from fixed samples: method
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validation using Ditylum brightwellii (T. West) Grunow. Diatom Research, 26: 43-49.
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Maier, M.A., and Peterson, T.D. (2014) Observations of a diatom chytrid parasite in the lower Columbia River.
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Northwest Sci. 88:234-245.
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Matsuki, Y., Isagi, Y., and Suyama, Y. (2007) The determination of multiple microsatellite genotypes and DNA
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sequences from a single pollen grain. Mol Ecol Notes 7: 194–198.
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Smit, E., Leeflang, P., Glandorf, B., van Elsas, J.D., and Wernars, K. (1999) Analysis of fungal diversity in the
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wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient
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gel electrophoresis. Appl Environ Microbiol 65: 2614–2621.
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Truett, G.E., Heeger, P., Mynatt, R.L., and Truett, A.A. (2000) Preparation of PCR-quality mouse genomic
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DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29: 52–54.
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Fig. SD2.1. Light micrographs of different life cycle stages of a chytrid strain of Zygorhizidium planktonicum
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infecting a colony of the diatom Asterionella formosa. A, a zoospore (zs). B and C, intact sporangia (is). D, an
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empty sporangium (es). Scale bar = 20µm.
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Fig. SD2.2. Comparison of the fractions of successfully analyzed samples of a single sporangium of the chytrid
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Z. planktonicum, which infected the diatom A. formosa, using two extraction methods (HS10, HS30) (A), three
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extraction methods (HS10, EL and TS) (B), and three DNA polymerases (KOD FX Neo, MightyAmp, Ex Taq
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HS) (C), and those of a zoospore, a single intact sporangium, 2 intact sporangia, and an empty sporangium of Z.
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planktonicum (D). Same letters on the top of each bar indicate groups that were not significantly different
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(p≥0.05, binomial generalized linear mixed model with Tukey's multiple comparison test). Ex Taq HS
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(Exp3-ET) was excluded from the statistical analysis because its fraction was zero. IDs in parentheses of the
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x-axis labels indicate experimental procedures listed in Table SD2.1.
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Table SD2.1. Summary of experimental procedures for DNA extraction and PCR analysis
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Experimental
Condition of Zygorhizidium
DNA extraction
DNA
Primer
procedure
planktonicum (ZS) or filed
procedure (see
polymerase
set
samples
Table SD2.2)
Exp1-HS10
One intact sporangium of ZS
HS10
KOD FX Neo
SI03/SI16
Exp1-HS30
One intact sporangium of ZS
HS30
KOD FX Neo
SI03/SI16
Exp2-HS10
One intact sporangium of ZS
HS10
KOD FX Neo
EF4/EF3
Exp2-EL
One intact sporangium of ZS
EL
KOD FX Neo
EF4/EF3
Exp2-TS
One intact sporangium of ZS
TS
KOD FX Neo
EF4/EF3
Exp3-MA
One intact sporangium of ZS
EL
Mighty Amp
EF4/EF3
Exp3-ET
One intact sporangium of ZS
EL
Ex Taq HS
EF4/EF3
Exp4-ZS
One zoospore of ZS
EL
KOD FX Neo
EF4/EF3
Exp4-2IS
Two intact sporangia of ZS
EL
KOD FX Neo
EF4/EF3
Exp4-ES
One empty sporangium of ZS
EL
KOD FX Neo
EF4/EF3
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Table SD2.2. DNA extraction procedures used in this study
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DNA extraction
Reagent
Heating regimen
Reference
(Temperature in ˚C/time
procedure
in min)
HS10
Alkaline lysis buffer *
96/10
Truett et al., 2000
HS30
Alkaline lysis buffer *
96/30
Truett et al., 2000
EL
Enzymatic lysis buffer
37/60,96/10
Matsuki et al., 2007
TS
TE
-80/5,96/10
Kamikawa et al., 2007
*
Requires addition of neutralizing buffer after heating.
158
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Table SD2.3. Summaries of Tukey multiple comparison tests for the statistical tests for experiments of method
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evaluations (Fig.SD2 and Table SD1) based on the generalized linear mixed model with a binomial error
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distribution.
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Experiment 1: Comparisons of DNA extraction methods (Exp1-HS10, Exp1-HS30)
Standard
Hypothesis
Coefficient
error
z value
P value
Exp1-HS10 – Exp1-HS30 == 0
-4.522e-06
4.563
0
1
164
165
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Experiment 2: Comparisons of DNA extraction methods (Exp2-HS10, Exp2-EL, Exp2-TS)
Standard
Hypothesis
Coefficient
error
z value
P value
Exp2-HS10 - Exp2-EL == 0
-7.154E-16
0.9058
0
1
Exp2-TS - Exp2-EL == 0
-3.412
0.9908
-3.444
0.00167
Exp2-TS - Exp2-HS10 == 0
-3.412
0.9908
-3.444
0.00160
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Experiment 3: Comparisons of DNA polymerases (Exp2-EL, Exp3- MA)
Standard
Hypothesis
Coefficient
error
z value
P value
Exp3-MA - Exp2-EL == 0
-0.9009
0.9902
-0.91
0.363
169
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Experiment 4: Comparisons of life cycle stages (Exp4-ZS, Exp2-EL, Exp4-2IS, Exp4-ES)
Standard
Hypothesis
Coefficient
error
z value
P value
Exp4-2IS - Exp2-EL == 0
-0.7189
0.9305
-0.773
0.8616
Exp4-ES - Exp2-EL == 0
-2.7818
1.1449
-2.43
0.0681
Exp4-ZS - Exp2-EL == 0
-4.46
1.5339
-2.908
0.0181
Exp4-ES - Exp4-2IS == 0
-2.0629
1.0296
-2.004
0.1791
Exp4-ZS - Exp4-2IS == 0
-3.7411
1.4336
-2.61
0.0426
Exp4-ZS - Exp4-ES == 0
-1.6782
1.261
-1.331
0.5319
171
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