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Supplementary Methods
Real-time PCR for quantification of PLS copy number. The PLS region from G.
etunicatum genomic DNA was amplified by PCR using Pol4 and Pol7 primers1. This
gave a fragment of 595 bp. The PCR product was purified and cloned into pGEM-T
vector (Promega, Switzerland). 40 PLS clones were then sequenced and analysed
(Vector NTI sotfware, UK). From the 40 sequences recovered, 6 different sequences
were found that matched PLS1 variants previously documented1. Another 2 different
PLS1 types were found that had not been previously described but were found by us
more than once. One other sequence matched to the PLS2 type1.
From these sequences, plus all the previously published PLS1 and 2 variants, we
designed primers that amplify a 212 bp fragment within a completely conserved region
of the 595 bp PLS1 and 2 sequences. The oligonucleotide sequences were: forward
primer (Pls2) 5’-GTATGGCTGCTTGGGGTTTAC, and reverse primer (Pls3) 5’GTGTCACCATAAATAACCTACAAG. An additional oligonucleotide probe was also
designed that hybridizes to the 212bp fragment in a completely conserved region. This
probe was labelled with FAM at the 5’ end and TAMRA at the 3’ end. The sequence of
the probe was: 5’-CAATGGAGGTGTGGAAATTACTTG. In the real-time PCR
reaction, the fluorescence of this probe was measured at each cycle at the annealing
phase of the reaction. The probe only fluoresces after it has hybridized to the target
DNA. All primers, and the probe, were synthesized by Microsynth, Switzerland. FAMreal-time PCR amplification was performed in 40 ml containing 1X qPCR Mastermix
(Eurogentec, Belgium), 0.5 M each primer, 0.25 M probe, and 10 l DNA at
different concentrations. The thermal cycling conditions were an initial step of 50°C for
2 min and 95 °C for 10 min, followed by 60 cycles of 95 °C for 15 s and 60 °C for 1
min. Fluorescence data were collected using the ABI PRISM 7000 Sequence Detection
System (SDS; Applied Biosystems). The SDS software then generated each real-time
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PCR profile after multi-componenting analysis by plotting the log of the change in
fluorescence (delta Rn) versus cycle number. The cycle threshold parameter (Ct) was
determined by the SDS software as the fractional cycle number. This indicates where
the fluorescence crosses an arbitrary threshold intersecting the signal curves in their
exponential phases.
In each experiment, plasmid DNA containing known amounts of the PLS insert was
used as template in real-time PCR as a standard. Two-fold serial dilutions (ranging
between 1115 and 4.3 pg) of the purified plasmid DNA were included in each
experiment to generate the standard curve. The real-time PCR on the plasmid DNA was
performed in five independent experiments (5 replicates, with a minimum of 12 and a
maximum of 19 dilutions per replicate). The Ct values were then plotted against the log
of the initial pGEM-T vector concentration containing the PLS insert. All replicates
offered very similar results producing standard curves with regression coefficients (R2)
> 0.99. This regression was then be used for predictions of PLS copy number for a
given Ct value. In the same experiments, two-fold serial dilutions of G. etunicatum
genomic DNA (ranging between 4350 and 21 pg) were included in each experiment to
generate the Ct values for nuclear DNA copy number estimation. Five independent
experiments were performed, each with 12 concentrations of the genomic DNA. All
replicates offered very similar results producing standard curves with regression
coefficients (R2) > 0.99. The number of copies of PLS could then be calculated for each
sample of target DNA, based on the regressions using the plasmid DNA as standards2.
From this we could then calculate the copy number of the PLS per 37.45 Mbases of
DNA; the amount of DNA in each G. etunicatum nucleus. As a control we used herring
sperm DNA as a carrier to yield 10 ng of total DNA per sample in a total volume of 40
l, irrespective of the dilution of the target DNA to show that the estimation of copy
number is not biased by the amount of non-target DNA added to the reaction.
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The DNA amplified in the quantitative PCR reactions was cloned and sequenced
to confirm that it was the PLS region and that we had amplified different variants of the
PLS region. All positive clones contained the PLS sequence. We found both the PLS1
and PLS2 sequence variants and additionally we found several variants of the PLS1
region. Because we designed the primers for quantitative PCR in order to amplify a
rather conserved region of the PLS (so that we would not amplify only specific types) it
is not possible for us to say what is the total number of variants that we have amplified.
We cannot say exactly how many of the different PLS1 sequences were amplified
although certainly several (in addition to the PLS2 type). This confirms that the number
of variants amplified in the quantitative PCR is considerably greater than the number of
copies that exist per nucleus, showing that the low copy number that we report for this
gene cannot be an artefact of amplification of only 1 or 2 of the variants.
Low copy number could potentially be an artefact of differences in amplification
efficiency between the genomic DNA preparations and the plasmid DNA that is used to
make the standard curve. In order to rule out this possibility, the amplification
efficiency from each sample must be 95% or more. Furthermore, it is essential that in
each replicate experiment, the efficiency of the amplification of the plasmid (used for
making the standard curve) is also over 95% efficient. The efficiency of amplification is
measured by the slope, with a slope of –3.3 being 100% efficiency3. R2 for the
regression of the amplification intensity for different concentrations of DNA must also
be ≥ 0.993. In our experiments, all reported values in the quantitative PCR met these
criteria. This means that the low copy number of the PLS region that we report cannot
be an artefact caused by differences in amplification efficiency.
We also performed two additional controls to check efficiency Firstly, to check
that the standard curve was not biased by using plasmid rather than linear DNA
molecules, we also repeated the experiment using amplified PCR product of the
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different cloned PLS1 and 2 variants mixed together. Secondly, we repeated the
experiment with the Sybr-green detection system that does not involve hybridization of
a fluorescent probe within the PCR reaction. Both of these experiments gave the same
result for the quantification of PLS copy number.
Analysis of re-association kinetics data. If a DNA molecule is melted and allowed to
re-associate, the complexity of the genome dictates the rate in which duplex DNA will
form. Renaturation, or duplex formation requires random collisions between two singlestranded molecules. This process follows second-order kinetics and is concentration
dependent. The C0t value is defined as the amount of time required for one-half of the
DNA to re-anneal or form duplex DNA. The units for this parameter is moles of
nucleotides per liter per second. The more complex the genome of interest, the longer it
will take for sequences to re-anneal. Consequently, the C0t value will be larger. EC0t
represents the equivalent C0t corrected for non-standard salt concentration, where C0t is
the product of the total DNA concentration C0 multiplied by the time in seconds t
during renaturation. Data of re-association kinetics were those of Hosny 19974. We reanalysed these data using the least-square program, as reported previously5,6. The
original calculation of the size of the haploid genome was made by manual estimation
on the plotted re-association kinetics data and was also based on C0t values4. The reanalysis was performed because the least-square program gives a more accurate
calculation of the haploid genome size than that calculated manually from the reassociation curve. Furthermore, we used EC0t values in the calculations because they
give a more precise measurement of genome size5,7. We have used the least squares
model for analysis because it can be used when only about 50% of the DNA has reannealed6, which is often the case when measuring re-association of large genomes such
as that of Scutellospora castanea.
References
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1.
Pawlowska, T. E. & Taylor, J. W. Organization of genetic variation in individuals
of arbuscular mycorrhizal fungi. Nature 427, 733-737. (2004).
2.
Alonso, A. et al. Real-time PCR designs to estimate nuclear and mitochondrial
DNA copy number in forensic and ancient DNA studies. Forensic Science
International 139, 141-149 (2004).
3.
Pfaffl, M. W. A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Research 29, e45 (2001).
4.
Hosny, M. GC content, genome size of Glomales. Genome complexity and
polymorphism of rDNA in Scutellospora castanea. Phd thesis, University of
Burdundy (1997).
5.
Hijri, M. & Sanders, I. R. The arbuscular mycorrhizal fungus Glomus
intraradicesis haploid and has a small genome size in the lower limit of
eukaryotes. Fungal Genetics & Biology 41, 253-261 (2004).
6.
Pearson, W. R., Davidson, E. H. & Britten, R. J. A program for least squares
analysis of reassociation and hybridization data. Nuceicl Acids Research 4, 1727–
1735. (1977).
7.
Britten, R. J., Graham, D.E. & Neufeld, B.R. Analysis of repeating DNA
sequences by reassociation. In: Grossman, E. L. & Moldave, K. (Eds.), Methods
in Enzymology, 29E. Academic Press, New York, pp. 363–418 (1974).