Basic HTML Version
International Journal of Marine Science 2014, Vol.4, No.50, 1-22
http://ijms.biopublisher.ca
14
Symbiodinium
rbcL
,
psI
(subunit III),
pgpase
,
nrt2,
and
apx1
, and host coral
actb
,
trp1
,
tuba
,
ezrin, cplap2
,
oatp
, and
trcc
. For the latter four genes, primers were
designed with CLC Main Workbench (ver. 6.8) from
published cDNA sequences (Mayfield et al., 2011).
Two additional genes,
Symbiodinium
and host coral
hsp70
were measured in Mayfield et al. (2011),
though the data were re-analyzed for the global site of
origin comparison described below. The 24 RNAs
were diluted to 20 ng μl
-1
, and 10 μl (200 ng) were
converted to cDNA with 1x Solaris RNA spike and
the High Capacity™ cDNA synthesis kit (Life
Technologies) according to the respective manufacturer’s
recommendations, precipitated with 0.1 vol sodium
acetate (3 M, pH 5.2) and 1 vol isopropanol, and
stored at -20
℃
. At a later date, cDNAs were
centrifuged at 12,000
xg
for 10 min at 4
℃
, washed
once with 75% ethanol, centrifuged at 8,000
xg
for 5
min, dried on the benchtop after decanting the ethanol,
and dissolved in 80 μl diethylpyrocarbonate-treated,
double-distilled water. Real-time PCRs (20 µl) were
conducted in triplicate on a StepOnePlus™ real-time
PCR machine (Life Technologies) with 1x (10 µl)
EZ-TIME™ SYBR® Green mastermix with ROX™
passive reference dye (Yeastern Biotech. Co., Ltd.),
the primer concentrations found in Table 4, and 2 μl
of the 4-fold-diluted cDNA. Serial dilutions of a
randomly selected cDNA sample were run on each
96-well plate to estimate the efficiency of each assay
(
sensu
Bower et al., 2007), all of which were shown to
be between 95-105%. Melt curves were conducted
after all reactions in order to verify the absence of
non-specific binding.
3.5 VTE: target gene expression
The expression of
Symbiodinium hsp70
and
nrt2
, as
well as host coral
actb
,
trp1
,
tuba
,
ezrin
,
cplap2
,
oatp
,
trcc
, and
hsp70
was assessed in the 24 samples of the
VTE sampled after seven days. Four additional genes;
rbcL
,
psI
(subunit III),
pgpase
, and
apx1
, were
measured in Mayfield et al. (2012a), though the data
were re-analyzed for the global site of origin
comparison described below. The SYBR Green
mastermix, reaction volumes, and real-time PCR
instrument described above were used. However, in
certain cases (noted in Table 4), slightly different
primer concentrations and thermocycling conditions
were used between samples of the two experiments.
3.6 Gene expression normalization
For both experiments, the expression of each gene was
normalized to Solaris spike recovery as described by
Putnam et al. (2013). Then, host and
Symbiodinium
spike-normalized gene expression values were divided
by the host and
Symbiodinium
GCPs, respectively; as
corals consist of two eukaryotes, both reverse
transcription efficiency and
biological composition
controls are necessary to generate accurate
macromolecular expression data (Mayfield et al.,
2013b, d). This circumvents the need for housekeeping
genes, which cannot be validated with confidence in
endosymbiotic organisms (Mayfield et al., 2009).
Because DNA samples were pooled and re-purified in
the ETE and re-extracted in the VTE, both host and
Symbiodinium
GCPs were recalculated with real-time
PCR as described by Mayfield et al. (2012a).
3.7
Symbiodinium
genotyping
Both previously published (Mayfield et al., 2012a)
and unpublished (Ruth D. Gates, personal
communication) works have found
S. hystrix
from
Southern Taiwan to predominantly possess
Symbiodinium
of clade C. However, as the former study used
restriction digests of PCR-amplified
18s
genes and the
latter used PCR of the
its2
marker followed by cloning
and sequencing, it is possible that background
Symbiodinium
haplotypes may have been overlooked.
Real-time PCR, which represents a more sensitive
means of detecting cryptic
Symbiodinium
diversity
(Mieog et al., 2007), was therefore utilized with all
DNAs from both experiments (
n
=48). Real-time
PCRs were conducted for the clade A, C, and D assays
of Correa et al. (2009), as these are thought to be the
dominant types found in corals of Southern Taiwan
(Chen et al., 2005). Reactions (20 µl) were conducted
with the mastermix described above for target gene
analysis with the primers and primer concentrations of
Correa et al. (2009). However, 20 ng of DNA were
used in each reaction. Due to the intragenomically
variable nature of the
its2
gene (Pochon et al., 2012),
these assays cannot be used to calculate exact ratios of
the dominant
Symbiodinium
types (e.g., 50% clade A
vs. 50% clade C) and are only suited for
presence/absence tests. “Presence” was defined
a