Supplementary Methods, Figures and Tables
This file contains supplementary methods and 5 supplementary figures (Figures S1 S5) and 1 supplementary table (Table S1).
Supplementary methods:
Determination of genetic relatedness and the choice of isolates to obtain a genetic
gradient
Without knowing the genetic system of the fungus it is not possible to set up precise
relatedness measures by measuring the number of generations that separate two
individuals from their last common ancestor. Consequently, we use a broad measure
of genetic relatedness, as used in many other studies, based on sequence differences
between pairs. The relatedness between each pair of isolates was determined by the
number of identical alleles at 13 loci. This was represented as a proportion, where 1
equals a different allele at all 13 loci and zero represents identical alleles at all 13 loci.
This was based on markers and AMF isolates described in Croll et al. 2008.
The choice of isolates was based on a collection of 18 genetically different R.
irregularis isolates that originate from the same field. Our aim was to obtain a
gradient of relatedness among pairs of the 18 isolates so that we could primarily use
regression analysis to test the hypothesis that genetic relatedness between isolates
influenced the variable measured (e.g. plant growth variables). Out of these pairs, we
also classified them into three categories containing equal numbers of different pairs.
1
Choice of pairs of isolates suitable for quantitative molecular analyses
A major constraint in choosing isolates out of the pool of 18 was to choose pairs that
could be distinguished by quantifying a small number of the 13 possible molecular
markers. Only a very small number of the markers can be used to distinguish between
genetically near pairs and not all markers were suitable for quantitative analyses.
The choice of the molecular approach used for estimating the relative proportion
of different AMF isolates in the roots of leek
This study looked at the relative proportion of pairs of genetically different isolates of
one AMF species in the roots of leek. It should be possible to quantify different coexisting AMF species or genera in the roots of plants using qPCR. However, when the
isolates are of the same species and sometimes genetically very closely related, such
an approach is not straightforward. There are very few markers available to
distinguish between these pairs of AMF isolates and none of them are suitable for
making specific probes to be used in conventional quantitative PCR (qPCR). The
approach we have used is a technique that was commonly employed in human and
animal quantitative genetics to assess allele frequency in mixed samples, before the
invention of qPCR. It is based on fluorescence intensity of amplicons measured in
capillary electrophoresis. Details about this technique are given in Angelard et al.
2010 and Ehinger et al. 2012. It is an end point PCR technique and cannot be used
alone to assess exact copy number but only relative proportions of different alleles.
However, if total AMF colonization is first measured with conventional qPCR giving
exact copy number, the proportions of each AMF isolate can then be converted into
copy number. We do not, however, recommend that exact copy numbers measured in
this study are compared with those from other studies, especially in other plant
2
species, unless: 1. The copy number of each marker of each AMF fungus used is
determined in advance and; 2. Quality of DNA extracted does not vary significantly
among the different plant species.
Checking colonization by AMF in Plantago lanceolata
The DNA of P. lanceolata was not considered to be of sufficient quality for
quantitative molecular tests. However, the DNA was of sufficient quality to use in
standard end-point PCR to amplify the AMF-specific markers described by Croll et
al. 2008. These amplifications revealed that the inoculated roots were colonized by
AMF with an amplification of DNA of the expected fragment size and that the
uninoculated plants were not colonized (no amplification). Several previous tests have
been conducted in our group with these primers and even in low stringency conditions
amplification of DNA has never been achieved from plant roots that were not
colonized by AMF. In addition, amplification of the markers was achieved from DNA
originating from colonized roots, even when the DNA quality was low. The primers
are described in Croll et al. 2008. The conditions for PCR were as in the methods
section of the main manuscript “Measurement of the relative proportion of different
AMF isolates in roots”.
Testing of quantitative PCR on the RAD15 gene as a reliable method for
quantifying the amount of AMF DNA present in a sample
We used quantitative PCR (q-PCR) to estimate the total colonisation by AMF in the
roots. Amplification of a given locus was previously shown to be correlated with the
number of spores and, thus, the amount of AMF DNA present in a sample (Gamper et
al 2008). We also tested this method by running q-PCR on nine DNA samples and
3
amplifying the RAD15 gene. This is a different target gene to the one used by
Gamper et al. 2008. These samples corresponded to extracts of known spore numbers,
ranging from 125 to 2000 spores. DNA was extracted from spores using the Cenis
method (Cenis 1992). There was a highly significant relationship between spore
number used for DNA extraction and the Ct value (Figure S2). We subsequently used
amplification of the RAD15 gene as a measure of AMF quantity in root samples. We
used this gene because it is present in equal copy number among several different R.
irregularis isolates from the same population (Corradi et al 2007). We also confirmed
that the copy number of RAD15 was the same among the isolates in this study (data
not shown). Thus, the RAD15 gene was considered a reliable marker for
quantification of R. irregularis in samples. The q-PCR mix for the tests contained: 0.4
µl of genomic DNA, 19.2µl of 2X Power Sybr® green (Applied Biosystems), 3.8 µl
of each of the two primers at concentration of 3 µM and 3.8 µl of water in a total
volume of 31µl. Quantitative PCR reactions were carried out on a Prism AB7900
quantitative PCR machine (Applied Biosystem). Thermal cycling conditions included
2 min at 50˚C, 10 min at 95˚C, followed by 45 cycles of 15 sec at 95˚C and 60 sec at
60˚C, then one cycle of 15 sec at 95˚C and 15 sec at 60˚C. Ct values were calculated
with the SDS2.4 software (Applied Biosystems).
Additional reference not cited in main text:
Cenis JL (1992). Rapid Extraction of Fungal DNA for Pcr Amplification. Nucleic
Acids Res 20: 2380-2380.
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Figure S1. Correlation between the variable “Phenotype” and the shoot dry mass at
the final harvest (week 16) for A. porrum (black circles) and P. lanceolata (white
circles). The correlation coefficients were highly significant (P < 0.001) for both A.
porrum (cor = 0.954) and P. lanceolata (cor = 0.963).
5
32
Ct value
30
28
26
24
2.2
2.4
2.6
2.8
3.0
3.2
Log Spore number
Figure S2. The relationship between the number of spores of R. irregularis and the Ct
values generated by q-PCR amplification of the RAD 15 gene. Each dot represents
one DNA extraction. Black dots represent DNA extracted from the isolate B2, gray
dots represent DNA extracted from isolate C5 and white dots represent DNA
extracted from the isolate C2. The line corresponds to the linear regression based on
lm model and tested by a Pearson correlation test (r = -0.982, p-value = 2.71 x 10-6).
6
Figure S3. (a) Shoot dry mass of P.lanceolata and (b) Shoot dry mass of A.porrum
after 16 weeks of growth with different mycorrhizal treatments. Bars represent means
± 1 SE. The black bar corresponds to the control treatment (NM), gray bars
correspond to the single inoculation treatments and white bars to the dual inoculation
treatments. Different letters above columns indicate a significant difference (p < 0.05)
7
according
to
Tukey-Kramer
8
(HSD)
test.
Figure S4. Box plot of total fungal colonization inside leek roots, as represented by 2ΔCt
values, in the different mycorrhizal treatments. The NM treatment is not shown as
no detectable DNA could be amplified from uninoculated roots. Horizontal lines in
the box plots represent medians and the others lines represent first and third quartiles.
Bars represent the range of variation including 90% of the values.
9
Figure S5. Frequency of the most abundant isolate in leek roots and the genetic
distance between the coexisting isolates. Each dot represents one experimental unit.
The line corresponds to the linear regression based on the lm model (F
R2 = 0.469, P ≤ 0.001).
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1, 102
= 91.91,
Table S1: Correlations between plant dry mass and the non-destructive plant
measurements at the final harvest (week 16).
Plant trait
df
Plant height
SPAD
Leaf thickness
Phenotype
182
182
182
182
Plant height
SPAD
Phenotype
188
184
188
t
Correlation
coefficient
P. lanceolata
2.563
0.193
4.851
0.338
2.405
0.176
48.265
0.963
A. porrum
12.716
0.680
4.531
0.317
43.993
0.954
11
P
< 0.01
< 0.001
< 0.05
< 0.001
< 0.001
< 0.001
< 0.001