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Single-cell PCR amplification of thecate dinoflagellates: a case study of Tripos

(Dinophyceae)

Article  in  Journal of Applied Phycology · April 2018

DOI: 10.1007/s10811-017-1269-1

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J Appl Phycol (2018) 30:1117–1124
DOI 10.1007/s10811-017-1269-1
Single-cell PCR amplification of thecate dinoflagellates: a case
study of Tripos (Dinophyceae)
A. Hernández-Rosas 1 & M. E. Meave del Castillo 2 & J. Díaz-Larrea 3 & F. Rodríguez 4
Received: 12 April 2017 / Revised and accepted: 4 September 2017 / Published online: 6 October 2017
# Springer Science+Business Media B.V. 2017
Abstract The relief of amplification inhibition in preserved
phytoplankton samples is a major challenge in genetic studies
relying on single-cell sequencing. The successful amplifica-
tion of rDNA genes varies considerably depending on the
organisms, fixatives, and time of analysis after collection.
Among other fixatives, RNAlater® is found to be a suitable
choice for PCR amplification after long-term storage. In this
study, we tested the performance of RNAlater® samples ap-
plied to single-cell PCR amplification in isolates of the thecate
dinoflagellate genus Tripos, with an amplification success
over 70%. The single-cell protocol proposed in this study
amplified an average of 650 pb of large subunit and small
subunit rDNA fragments in RNAlater® preserved cells after
5 months. Furthermore, it was possible to obtain rDNA se-
quences from samples up to 8 months old. The approach de-
scribed here could also be useful to amplify a wide range of
thecate and non-thecate dinoflagellate taxa, especially those
species difficult to maintain in culture.
* A. Hernández-Rosas
adrishrosas@gmail.com
1
Doctorado en Ciencias Biológicas y de la Salud, Universidad
Autónoma Metropolitana, Unidad Iztapalapa (UAM-I), Av. San
Rafael Atlixco #86, Col. Vicentina, Del. Iztapalapa Cd.,
09340 Mexico City, Mexico
2
Laboratorio de Fitoplancton Marino y Salobre, Departamento de
Hidrobiología, División de Ciencias Biológicas y de la Salud,
UAM-I, Mexico City, Mexico
3
Laboratorio de Macroalgas, Departamento de Hidrobiología,
División de Ciencias Biológicas y de la Salud, UAM-I, Mexico
City, Mexico
4
Instituto Oceanográfico de Vigo, IEO, Subida a radio Faro, 50,
36390 Vigo, Spain
Keywords RNAlater® . Tripos . LSU . SSU . rDNA
amplification
Introduction
Specific recognition of dinoflagellate taxa based on mor-
phological characters is not always feasible due to their sim-
ilarity at an intrageneric level (Reguera et al. 2011; Hansen
et al. 2000). This poses a relevant problem especially in the
case of harmful algal blooms (HABs) and toxic events,
which draw much attention due to their potential socioeco-
nomic impact in aquaculture resources and marine ecosys-
tems (Reguera 2002; Hallegraeff 2004). Therefore, a lot of
effort has been devoted to improve the characterization of
HAB species in multidisciplinary studies focusing on their
taxonomy, ecophysiology, and toxicology (Anderson et al.
2012; Fraga 2014).
In recent decades, the advent of molecular techniques
allowed to describe cryptic and pseudocryptic dinoflagel-
late species, generally based on rDNA gene sequencing.
However, the limited amount of genetic material, especially
in the case of these organisms that is difficult to maintain in
the laboratory, represents a major obstacle in some studies
(Andersen 2005; Graham and Wilcox 2000). Therefore, a
number of molecular studies using different thecate dino-
flagellate taxa (Marin et al. 2001; Godhe et al. 2002;
Auinger et al. 2008) have examined the PCR amplification
efficiencies on preserved samples using the most common
substances, such as formalin, ethanol, methanol, Lugol’s
solution, and formalin/methanol, with mixed success de-
pending on the particular organisms and protocols.
Specifically, the formalin/methanol mixture appears to
strongly inhibit PCR amplification, whereas other fixatives
such as Lugol’s iodine solution and ethanol do allow direct
1118
PCR on single cells following a recommended protocol
(Godhe et al. 2002). DNA alteration over time and the im-
portance of choosing an adequate preserving agent has been
identified in many cases as a crucial step, especially when
controlled temperature conditions and safe storage during
transport to the laboratory are not feasible (Godhe et al.
2002). In addition, the rigid wall on thecate dinoflagellates
can be an obstacle in the success of DNA extraction and the
steps used in the different techniques increase the chances
of cross-contamination (Ki and Han 2007).
Thecate dinoflagellates of the genus Tripos are cosmo-
politan and diverse marine organisms, with up to 155 spe-
cies (Guiry and Guiry 2017), commonly found in coastal
waters, although some species live in the open ocean. One
important characteristic is the autotomy phenomenon of
Tripos species; these species can cast off their horns be-
cause they do not need to have large horns for them to be
suspended on a sea column (Kofoid 1908). Their morpho-
logical variability at the intraspecific level and the overlap
in meristic characters hinder the identification of several
species and intraspecific categories, such as varieties and
forms (Sournia 1968; Taylor 1976; Balech 1988). When
considering these categories, the number of currently ac-
cepted taxonomic entities in the genus Tripos rises to 450
(Gómez 2013). Yet, such variability has only been explored
based on a few morphometric studies (López 1955;
Yarranton 1967; Hansen et al. 2000; Hernández Rosas
2011; Lyakh and Bryantseva 2014) that do not achieve un-
ambiguous specific resolution in all cases. Although spe-
cies of genus Tripos are non-toxic, they can proliferate with
damage to the ecosystem (Orellana et al. 2004; Morton
et al. 2011; Pichen and Probyn et al. 2011).
Molecular characterization of Tripos spp. based on single
cells and cultures would be very useful to delineate the phy-
logenetic position of species identified solely by morpholog-
ical means and to determine the putative presence of cryptic
and pseudocryptic species in this genus. Nonetheless, the ef-
ficiency of PCR amplifications either on live or preserved
single cells can be highly variable due to the inherent limita-
tions of this approach (small amount of template DNA, PCR
inhibition associated with cellular constituents or fixatives)
(Godhe et al. 2002; Ki et al. 2005; Auinger et al. 2008; Lang
and Kaczmarska 2011). These factors must be considered
when using cells from the natural environment in the amplifi-
cation protocol.
The objective of this study was to compare the suitability
of different fixatives on single-cell PCR amplification of
large subunit (LSU) rDNA in specimens of the dinoflagellate
genus Tripos, considering cases in which it is not possible to
transport living material to the laboratory. Specifically, we
tested the PCR success of (fresh, RNAlater®) preserved
specimens and common fixatives such as formalin and
Lugol’s iodine solution.
J Appl Phycol (2018) 30:1117–1124
Materials and methods
Sample collection and preservation Samples were collected
in Acapulco Bay, in the Tropical Mexican Pacific (99° 50′
52″–99° 56′ N and 16° 47′–16° 51′ 40″ W), and in the Ría
de Vigo, NW Spain (42° 11′ 56.46″ N and 8° 47′ 54.09″ W),
with a 60-μm mesh size net and a Van Dorn bottle. Twenty-six
samples were preserved in four ways: (1) 4% formalin fixa-
tion. (2) Lugol fixation: 1 mL Lugol’s iodine solution was
added to a bottle sample of 250 mL, with a storage time of
2 months. (3) Live samples obtained from the bottle and the
net kept at a low temperature (4 °C). Individual cells were
isolated, placed in PCR tubes, and stored at − 20 °C for later
amplification. (4) RNAlater®: Samples obtained from the
bottle were concentrated by reverse filtration. Net live samples
were placed in sterile bottles; two drops of Lugol’s iodine
were added, and samples stored at 4 °C. Then each sample
was allowed to settle by positive gravity for 4 h. The concen-
trate was placed in cryogenic vials or Eppendorf tubes and
RNAlater® was added in a 1:6 ratio (Ambion 2010). The
tubes were stored at 4 °C until analysis.
Cell isolation Three hundred sixty cells of the genus Tripos
were isolated using a micropipette made from a capillary
thinned on a Bunsen burner (Fireboy, Integra Biosciences
AG, CH) of the four ways of preservation. They were washed
in three drops of Milli-Q water to eliminate possible contam-
inants and placed in PCR tubes, each of those containing a
single cell. TE buffer (1 μL) was added to Lugol-fixed sam-
ples and stored at − 80 °C with RNAlater® in order to avoid
DNA degradation. The use of TE buffer also had the advan-
tage of preventing cell disruption, commonly observed in
Milli-Q water due to osmotic shock. Photomicrographs were
taken of each isolated cell, and species identification was con-
firmed by morphological features as silhouette, dimensions,
shape of apical and antapical horns, and structures as ribs on
thecae. Lastly, the samples were stored at − 80 °C.
Cell DNA extraction and amplification Five procedures
were used for DNA extraction on preserved samples accord-
ing to the four methods, formerly described: (1) the plant/
fungi gDNA (Nzytech) extraction kit was used following the
manufacturer’s recommendations; (2) Ten percent (Sigma) of
Chelex was used following the protocol by Richlen and
Barber (2005); (3) temperature thermal shock: samples were
frozen at − 80 °C and heated at 95 °C for 6 min (Saldarriaga
et al. 2004); (4) incubation with proteinase K (200 μg mL−1) at
55 °C for 50 min (Ki et al. 2005); and (5) sonication: PCR
tubes were placed in an ultrasonic bath for 5–10 min and ice
was added to the water bath to avoid heating.
The LSU and small subunit (SSU) rDNA regions were
amplified using the primers and combinations shown in
Table 1 and Fig. 1. D1–D2 sequences of LSU rDNA were
J Appl Phycol (2018) 30:1117–1124 1119

Table 1 Primers and their

combination used in the protocols Region Primers Reference Combination of primers Fragment
for amplification of the LSU and
SSU rDNA LSU D1R-F Scholin et al. D1R-F vs D2C-R D1–D2
D1C-R Scholin et al. D1R-F vs D1C-R D1
D2C-R Scholin et al. D1R-F vs D3B-R D1–D3
D2Ra-F Scholin et al. D2Ra-F vs D2C-R D2
D3B-R Scholin et al. D2Ra-F vs D3B-R D2–D3
SSU SR1-F Matsuoka et al. SR1-F vs SR9- R SR1–SR9
SR4-F Matsuoka et al. SR1-F vs SR7-R SR1–SR7
SR7-R Matsuoka et al. SR4-F vs SR7-R SR4–SR7
SR9-R Matsuoka et al. SR4-F vs SR9-R SR4–SR9

assembled from D1- and D2-amplified fragments from differ-
ent PCR tubes containing individuals of the same morphospe-
cies. PCR reaction mixtures (25 μL) contained the following
components: Milli-Q water, 2UTaq DNA polymerase or
2UPaq5000 DNA polymerase, 10× buffer, 10 mM dNTPs,
10 μmol of each primer, and bovine serum albumin (BSA)
(Sigma-Aldrich). Alternatively, samples were amplified using
Maxima Hot Start PCR Master Mix (2X) (Thermo Scientific).
Two different Taq polymerases were assayed: Taq DNA po-
lymerase (Qiagen) and Paq5000 DNA polymerase (Agilent
Technologies). In the case of the Maxima Hot Start, a 50 μL +
10 μmol mixture of each primer was used. The conditions of
amplification used for each region are detailed in Tables 2 and
3. The PCR products were observed in a 1% agarose gel, and
the amplicons were purified using ExoSAP-IT® (USB) ac-
cording to the manufacturer’s indications. The purified PCR
products were stored at − 20 °C until sequencing.
Sequencing and aligning of sequences Sequencing was per-
formed using an AB 3130 sequencer (Applied Biosystems) in
the CACTI (Centro de Apoyo Científico y Tecnológico a la
Investigación, University of Vigo, Spain). The sequences ob-
tained were aligned using the BioEdit program (version 7.2.5)
and were compared with sequences in the GenBank using the
BLAST (Basic Local Alignment Search Tool) program.
Genetic results were correlated with morphological features
from photomicrographs taken during cell isolation on the
same organisms (or those sharing identical characters to the
sample used in the PCR amplification).
Results and discussion
Cell preservation and isolation RNAlater® has been used
mainly to preserve tissue fragments of mammals, fish, am-
phibians, plants, and bacteria films (Gorokhova 2005,
Ambion 2010, Gray et al. 2011, Hamilton 2015). In the pres-
ent study, the use of RNAlater® to preserve genomic DNA of
the thecate dinoflagellate genus Tripos enabled the successful
PCR amplification of partial sequences from rDNA genes.
One of the most important advantages of using RNAlater®
is that it allowed us to preserve nucleic acids immediately after
sample collection at ambient temperature. The manufacturer
states that tissues embedded in RNAlater® may be stored at
− 20 °C indefinitely or at 4 °C for 1 month, at 25 °C for 1 week,
and at 37 °C for 1 day. RNAlater® is also simpler and easier
to use compared with compounds such as ethanol, methanol,
and the formalin/methanol mixture (Marin et al. 2001; Godhe
et al. 2002) that needs to be stored immediately after collection
at low temperature. The choice of a suitable fixative for geno-
mic analyses also varies depending on the studied organism.
Gorokhova (2005) stated that the storage time of Artemia

Fig. 1 a, b TIFF. DNAr

fragments and position of the
primers used in the amplification
protocols. a Dominions of the
LSU region. b Fragments of the
SSU region. Forward 3′
(rightwards arrow), reverse 5′
(leftwards arrow)
1120 J Appl Phycol (2018) 30:1117–1124

Table 2 Amplification conditions for the D1–D3 dominions of the results of the present study, we recommend a maximum
LSU region, using Taq DNA polymerase (Qiagen), Paq5000 DNA
storage time of 5 months in samples used for single-cell
polymerase (Agilent Technologies), and Maxima Hot Start PCR Master
Mix (2X) (Thermo Scientific). Amplification conditions were the same in PCR to avoid morphological alterations and a decrease in
both Taq DNA polymerase and Paq5000 DNA polymerase the amplification success and when the difficulty is related
to the rapidity of transporting live sample to the laboratory
Taq-Paq5000 DNA Pol Maxima Hot Start PCR Master Mix
where the amplification is to be carried out.
°C min °C min In this regard, no amplification was obtained in the PCR
reactions when the cell consistency was lost and the release of
1 94 3 35 cycles 95 4 35 cycles the nucleus was confirmed (Fig. 2c, d). This degradation
2 94 1 95 30 s
might be due also to excessive time during sample handling.
3 50–58a 1 Tmb 30 s
However, cells became much less fragile when stored in
4 72 3 72 1
RNAlater® solution; natural phytoplankton samples usually
5 72 10 72 10
contain a variety of organisms and manual isolation of target
6 4 ∞ 4 ∞ cells, Tripos in the case of the present study, needs to be done
1. Initial denaturation, 2. Denaturation, 3. Hybridization, 4. Extension, 5. as fast as possible to avoid cell damage that will difficult
Final extension, 6. Sample maintenance further genetic analyses. This is particularly important in the
a
T° hybridization to amplify the DNAr fragments (Table 1) case of large cells such as those of the genus Tripos that pos-
b
Average primer temperature sess a wide surface and ornamental structures that easily attach
other biological materials, especially in those samples with
high densities of organisms and/or detritus (Fig. 2d). In some
nauplii with RNAlater® could be extended up to 8 months at cases, the proper isolation of clean individuals of the genus
4 °C while the recommended time is only 1 month according Tripos was impossible and contaminant nucleic acids from
to the manufacturer’s instructions. However, cells used in other dinoflagellates or planktonic organisms, such as cope-
PCR amplifications stored up to 5 months at 4 °C were also pods, were amplified instead.
positive in the present study (Table 4). As mentioned previ- For these reasons, it is recommended to take small aliquots
ously, the original morphology of Tripos was maintained in to isolate individual cells, avoiding an extended exposure of
good condition after that period with no apparent signs of cell samples under the microscope. After isolation, individual cells
rupture, as corroborated by their observations under the light must be frozen at − 80 °C and it is highly recommended to
microscope. These cells showed a well-consolidated nucle- process them in the shortest delays (less than 10 days) (Fig. 4).
us (Fig. 2a, b), a characteristic that according to Echeverría
et al. (1993) indicates that the DNA did not undergo degra-
Extraction The DNA extraction protocols, based on a com-
dation. Amplifications carried out using these preserved
mercial kit (plant/fungi gDNA Nzytech) and the Chelex pro-
cells showed up ~ 80% of success. Thus, based in the
cedure, were not successful for further PCR amplifications of
rDNA genes in single cells of Tripos. The greatest drawback
Table 3 Amplification conditions for the fragments of the SSU region, in using these protocols was that they required multiple steps
using Taq DNA polymerase (Qiagen), Paq5000 DNA polymerase when transferring materials for DNA extraction, increasing
(Agilent Technologies), and Maxima Hot Start PCR Master Mix (2X) the probability to lose or contaminate the DNA. In contrast,
(Thermo Scientific). Amplification conditions were the same in both
Taq DNA polymerase and Paq5000 DNA polymerase
the use of thermal shock (− 80 to 95 °C) and incubation with
proteinase K proved to be both adequate methods for DNA
Taq-Paq5000 DNA Pol Maxima Hot Start PCR Master Mix extraction. The advantage of these methods lied in the fact that
the permeation of cell structure takes place in the same PCR
°C min °C min
tube where the organisms were isolated and further amplified.
1 95 3 35 cycles 95 4 35 cycles The use of an ultrasonic bath to disrupt cells prior to PCR
2 95 1 95 30 s did not render any particular advantage; for single cells, this is
3 56–58a 1 Tmb 30 s not always effective, particularly when working with cells
4 72 3 72 1 with a thick theca. The Tripos cells that were exposed to ul-
5 72 10 72 10 trasonic method, even for up to 10 min, did not show cell wall
6 4 ∞ 4 ∞ fragmentation and did not release their genetic contents,
resulting in difficulty in further PCR amplification.
1. Initial denaturation, 2. Denaturation, 3. Hybridization, 4. Extension, 5.
Final extension, 6. Sample maintenance
a
T° hybridization to amplify the DNAr fragments (Table 1) Amplification Successful amplifications were only obtained
b
Average primer temperature with the combinations of primers D1R-F vs D1C-R (D1) and
J Appl Phycol (2018) 30:1117–1124 1121

Table 4 Amplified LSU and SSU sequences obtained from a single Tripos cell. Unicellular organisms of Tripos from Acapulco Bay, Mexico, were
preserved in RNAlater® and those from Ria de Vigo, Spain, were fresh cells. The organisms were identified by morphological characteristics. *Cells
with more than 5 months in RNAlater®. a, b, and c correspond to sequences of different cells, identified as the same species from the same sample

Organism Locality rADN No. access GenBank

T. balechii f. balechii* (Meave del Castillo, Acapulco Bay, Mexico LSU (D1–D2) MF927991
Okolodkov, and Zamudio) Gómez
T. balechii f. balechii (a) Acapulco Bay, Mexico LSU (D1) MF927992
T. balechii f. balechii (b) Acapulco Bay, Mexico LSU (D1) MF927993
T. balechii f. balechii (c) Acapulco Bay, Mexico LSU (D1) MF927994
T. muelleri f. parallelus* (=Ceratium breve var. parallelum Acapulco Bay, Mexico LSU (D1–D2) MF927995
(Schmidt) Jorgensen) (Schmidt) Gómez
T. deflexus* (Kofoid) Gómez Acapulco Bay, Mexico LSU (D1–D2) MF927996
T. furca var. furca (Ehrenberg) Gómez Acapulco Bay, Mexico LSU (D1) MF927997
T. furca var. furca Ria de Vigo, Spain LSU (D1) MF927998
T. fusus (Ehrenberg) Gómez Acapulco Bay, Mexico LSU (D1–D2) MF927999
T. fusus Ria de Vigo, Spain LSU (D1–D2) MF928000
T. massiliensis var. armatus* (Karsten) Gómez (a) Acapulco Bay, Mexico LSU (D1) MF928001
T. massiliensis var. armatus (b) Acapulco Bay, Mexico LSU (D1) MF928002
T. muelleri var. atlanticus (Ostenfeld) Gómez Ria de Vigo, Spain LSU (D1–D2) MF928003
T. balechii f. balechii Acapulco Bay, Mexico SSU MF927973
T. muelleri f. parallelus (a) Acapulco Bay, Mexico SSU MF927974
T. muelleri f. parallelus (b) Acapulco Bay, Mexico SSU MF927975
T. candelabrus (Ehrenberg) Gómez Acapulco Bay, Mexico SSU MF927976
T. candelabrus Ria de Vigo, Spain SSU MF927977
T. deflexus* Acapulco Bay, Mexico SSU MF927978
T. falcatus (Kofoid) Gómez Acapulco Bay, Mexico SSU MF927979
T. furca var. eugrammus* (Erhenberg) Gómez Acapulco Bay, Mexico SSU MF927980
T. furca var. furca Acapulco Bay, Mexico SSU MF927981
T. furca var. furca (a) Ria de Vigo, Spain SSU MF927982
T. furca var. furca (b) Ria de Vigo, Spain SSU MF927983
T. fusus (a) Acapulco Bay, Mexico SSU MF927984
T. fusus (b) Acapulco Bay, Mexico SSU MF927985
T. fusus Ria de Vigo, Spain SSU MF927986
T. massiliensis var. armatus* Acapulco Bay, Mexico SSU MF927989
T. massiliensis var. armatus Ria de Vigo, Spain SSU MF927988
T. muelleri var. atlanticus Ria de Vigo, Spain SSU MF927990

D2Ra-F vs D2C-R (D2) of LSU rDNA and SR4-F vs SR9-R
of SSU rDNA genes, respectively.
The first PCR reactions on samples stored for month in
Lugol’s solution, formalin, and ethanol were negative in all
cases. Their failure coincided with observations by Marin
et al. (2001) who stated that some purification steps were
previously required in samples of dinoflagellate cells, as the
thecate genus Dinophysis. In addition, PCR trials on cells
isolated from a fresh sample and immediately frozen at
− 80 °C (without fixatives) were only 20% successful.
The use of TE buffer allowed positive PCR amplifications
in fresh cells, especially from NW Spain, but not in previ-
ously fixed cells from Mexico. The low rates of success in
the latter samples could be due to their inappropriate han-
dling due to the long period elapsed (3–4 days) at high
temperature (> 20 °C) between their collection and the ar-
rival at the laboratory.
The use of Taq polymerase and Maxima Hot Start PCR
Master Mix, exclusively with cells preserved in RNAlater®,
provided similar amplification success and readable final se-
quences (Fig. 3a, b). However, the use of the Master Mix was
nearly always effective, with a rate of 70% (32 positives out of
46 samples).
The addition of BSA protein to the PCR reaction mixtures
is advised to prevent the adhesion of enzymes to the reaction
tubes (Farell and Alexandre 2012). Therefore, we added three
1122 J Appl Phycol (2018) 30:1117–1124

Fig. 2 a–d TIFF. Tripos

unicellular organisms used in the
PCR, stained with SYBR Green
to observe the nuclei. a
T. muelleri. b–d T. furca. a, b
Whole cells with defined
morphology and nuclei that
proved positive in the PCR
amplification. c, d Damaged cells
with material adhered to the
periphery that proved negative in
the amplification

different volumes of BSA (0.5, 0.75, and 1 μL) to the PCR
reaction mixture, to explore its effect on the amplification
success. Positive amplifications were obtained with 0.75 and
0.5 μL of BSA. This could be explained by a larger amount of
BSA required in order to increase the PCR yield on low purity
templates (Farell and Alexandre 2012).
Tripos cells preserved in RNAlater® that showed up neg-
ative results could be observed in many cases to remain intact
in the original tube after PCR cycling. When working with
cysts and vegetative cells of the thecate dinoflagellate genus
Protoperidinium, Matsuoka et al. (2006) found an alternative
extraction method. They recommend breaking the cell wall on
a glass slide with a sharp glass stick and then placing the
cytoplasm in a PCR tube. This must be done quickly, and
TE buffer must be added, since the genetic material is more
exposed to degradation with this technique (Fig. 4).

Fig. 3 a, b TIFF. PCR products

of the LSU and SSU obtained
from a Tripos cell preserved in
RNAlater®. Well-defined bands
of fragments larger than 600 pb
for the SSU region (primers SR4-
F vs SR9-R) and of 300 pb for D1
dominion (D1R-F vs D1C-R) of
LSU region. a Amplification
using Maxima Hot Start PCR
Master Mix. b Amplification
using the traditional Taq
polymerase mixture
J Appl Phycol (2018) 30:1117–1124
Fig. 4 TIFF. Protocol for sampling, preserving, revising, and PCR for
cells of Tripos by four methods of preserve and five procedures before
PCR amplification. In case of RNAlater®, vital steps to add the
preservative, storage at 4 °C, time minimum to examine aliquots,
transfer of the clean cell from Milli-Q water to PCR tubes, and freezing
of cells
Purification All the PCR products observed in agarose gel
with sizes from 300 to 400 pb (D1 and D2 domains of LSU)
and from 600 to 700 pb (fragment SR4-F vs SR7-R of SSU)
were purified with ExoSAP-IT®. Other purification methods,
commercial kits, led to a loss of DNA material that prevented
further sequencing.
1123
Sequencing The identity of the amplified LSU and SSU
rDNA sequences from Tripos cells were confirmed by their
comparison with other available records in GenBank.
Amplifications of partial LSU rDNA from different cells shar-
ing the same morphological features were obtained. D1 do-
main was easier to obtain than D2; this is the reason in the
difference of base pairs in the LSU region (Table 4). In the
case of the SSU rDNA region amplified, an average of 695 bp
were obtained for 13 species of Tripos, while an average of
690 bp were obtained for the LSU rDNA fragment (D1–D2
region) for six species of Tripos. In Tripos furca and
T. massiliensis, only the D1 domain of LSU rDNA could be
amplified (Table 4).
In conclusion, our results showed that cells of Tripos em-
bedded in RNAlater® could be stored up to 5 months, unlike
those preserved in Lugol’s iodine solution and formalin,
which never rendered positive DNA amplifications.
RNAlater® preserved intact cells and their morphological
features as well. The use of cells preserved with RNAlater®
for the amplification of the LSU and SSU rDNA regions led to
positive results in 50–70% of the samples assayed in this
study.
The protocol recommended in the present study provides a
simpler and faster way of preserving phytoplankton cells and
running successful PCR analyses, while preserving Tripos
morphological features for taxonomic analysis. This protocol
could also be used and adapted in the case of other thecate
dinoflagellates, unarmored dinoflagellates, or diatoms
(Hamilton et al. 2015).
Handling the cells at low temperatures and minimizing
their manipulation during the extraction procedure appear also
as relevant factors to achieve successful DNA sequencing of
marker regions like the ribosomal genes considered in the
present study.
Funding This research is part of the first author’s PhD dissertation at
the División de Ciencias Biológicas y de la Salud of the Universidad
Autónoma Metropolitana, Unidad Iztapalapa (UAM-I). The first author
had a Consejo Nacional de Ciencia y Tecnología (CONACyT) PhD
Scholarship: 174843. This study was funded too by the UAM project
titled BTaxonomía y biogeografía del fitoplancton marino y salobre en
costas mexicanas, con énfasis en el Pacífico Tropical Mexicano y porción
sur del Golfo de México.^
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