Fróðskaparrit - 01.07.2004, Blaðsíða 34

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HABITAT PREFERENCES OF THE ARBUSCULAR MYCORRHIZAL FUNGI
OF THE FAROES COMPARED WITH OTHER PLACES
Field work
During the summer 2002, 3 cores of soil,
approx 5-6 cm wide and 8 cm deep with
Agrostis capillaris were taken from each
site at each sampling day. The roots were
washed, dried and stored at room tempera-
ture until extraction.
The data are based upon two sampling
dates for each site. Three plant roots were
sampled and the DNA is extracted from a
pool of the three roots. From one of the low
altitude south-facing sites no PCR product
was obtained from one sampling date,
while from the other low altitude south-fac-
ing site the cloning was not very successful,
providing only 3 clones. Therefore the
north-facing sites might be overrepresented
(Table 1).
For the lab grown A. capillaris 8 samples
from Sourhope and 8 from Sornfelli were
taken, but only 4 from Velbastað. The suc-
cess rate for the DNA amplifícations was
50% for Sornfelli, 75% for Velbastað and
100% for Sourhope.
DNA extraction
The DNA was extracted by the CTAB
method (Edwards et ai, 1997). Roots were
ground in liquid nitrogen, and then the
DNA was extracted by phenol and chloro-
form. After washing with ethanol the ex-
tract went through an extra purification
with StrataPrep PCR Purifícation Kit from
Stratagene. These purifícations are neces-
sary to get rid of inhibitors.
The DNA from the samples used in the
T-RLFP analysis was extracted with the Qi-
agen DNeasy Plant Mini Kit according to
the manufacturer’s protocol.
PCR
The genes here amplified with PCR are
those that defíne the ribosomal RNA
(rRNA). For this purpose TAQ polymerase
(Invitrogen) was used together with the
primers NS31 (Simon et al., 1992), AMl
(Helgason et al., 1998) and AM2 (Ridg-
way, unpubl.). NS31 is an general primer,
valid for all eucaryotes, while AMl is a
specific primer, amplifying especially glo-
malean fungal types, but not paraglomalean
and Archaeospora types. AM2 amplifies a
shorter segment and a wider range of glo-
malean fungi, paraglomalean and Ar-
chaeospora types. The drawback of using
AM2 is that more ascomycetes are ampli-
fíed together with the glomalean fungi.
The reactions were performed using 0.2
mM dNTPs, 1 Opmol of each primer and the
supplied reaction buffer to a fínal volume of
50 pL. The PCR-amplification was done on
a PTC-200 machine (MJ Research), 94°C
for 3 min, then 30 cycles of 94°C for 1 min,
58°C for 1 min and 72°C for 1 min. The fí-
nal step was 10 min. at 72°C.
The T-RLFP samples were amplified us-
ing Qiagen Multiplex PCR Kit, 5 pmol of
each primer (AMI and NS31). Q-solution,
a reagent modyfying the melting behaviour
of DNA (Qiagen), was added according to
the manufacturer’s protocol. Amplifícation
on a PTC-200 machine, 95°C for 15 min.,
40 cycles with 94°C for 30 sec., 58°C for
90 sec. and 72°C for 90 sec. The fínal ex-
tension was 72°C for 10 min.
Cloning
The PCR product was inserted into a
pGEM-T vector (Promega), and then the