Páginas 000-000
IDESIA (Chile), 2018
Rapid diagnostic PCR method for identification of the genera
Sarcocornia and Salicornia.
Método de PCR de diagnóstico rápido para identificación de los géneros
Sarcocornia y Salicornia.
Roberto Contreras1*, Bernardo Sepúlveda1, Fernanda Aguayo1, Vincenzo Porcile1
ABSTRACT
Plants that belong to different genera sometimes may present close morphological similarity and cannot be distinguished phenotypically by non-specialists. The aim of this study was to develop a simple diagnostic PCR for the identification of plants of
Sarcocornia and Salicornia and to test this new procedure to identify 82 samples of Sarcocornia neii from coastal and valleys of
the Atacama region of Chile. Six primer pairs were designed from ETS sequences of the genera Sarcocornia and Salicornia and
evaluated for the identification of both genera. Primers with a mismatch in the 3’ nucleotide indicate the site of the SNP. Four primer
pairs (SALI2F-2R, SALI3F-4R, SARCO1F-1R and SARCO3F-3R) were selected to develop an efficient and simple diagnostic
PCR for the identification of Sarcocornia and Salicornia. The results show that with this method is possible to identify Sarcocornia
and Salicornia. This method may be useful as an approach for genetic traceability of conserved products (sea asparagus). This
work provides an applicable and efficient method using only DNA, PCR and electrophoresis.
Key words: Sarcocornia, PCR, allele-specific primer, SNP.
RESUMEN
Las plantas que pertenecen a diferentes géneros, a veces, pueden presentar fuertes similitudes morfológicas y pueden no ser
identificadas fenotípicamente por no especialistas. El objetivo de este trabajo fue desarrollar una PCR diagnóstico para la
identificación de plantas de los géneros Sarcocornia y Salicornia y probar este nuevo procedimiento para identificar 82 muestras
de Sarcocornia neii distribuidas en la costa y valles de la región de Atacama. Se diseñó seis parejas de cebadores a partir de
secuencias ETS de los géneros Sarcocornia y Salicornia, y se evaluó la identificación de cada género. Los cebadores contienen un
desajuste en el nucleótido 3’ que corresponde al sitio del SNP. Se seleccionaron cuatro pares de cebadores (SALI2F-2R, SALI3F-4R,
SARCO1F-1R y SARCO3F-3R) para desarrollar una PCR de diagnóstico eficiente y simple para la identificación de los géneros
Sarcocornia y Salicornia. El resultado muestra que con este método se puede identificar los géneros Sarcocornia y Salicornia.
Adicionalmente, este método puede ser útil como propuesta para la trazabilidad genética de productos conservados (espárragos
de mar). Este trabajo proporciona una aplicación eficiente, utilizando sólo ADN, PCR y electroforesis.
Palabras clave: Sarcocornia, PCR, primer específicos de alelos, SNP.
Introduction
The genus Sarcocornia was established by Scott
(1978), who separated it from Salicornia L. and
Arthrocnemum Moq on the basis of morphological
characteristics. Currently, Sarcocornia includes
about 28 species of perennial succulent halophytes
distributed worldwide (Steffen et al., 2015). Since
Scott’s publication, the validity of the distinction of
1
*
these genera has been questioned. Taxonomic studies
of Salicornia in South America initially indicated
the presence of this genus in Chile (Gunckel, 1978)
and Peru (Gutte and Muller, 1985) as well as other
countries; however, Scott (1978) concluded that
these plants belong to the genus Sarcocornia. Scott
(1978) defined two species in the South American
continent as Sarcocornia fruticosa and Sarcocornia
pulvinata. Later, Alonso and Crespo (2008) redefined
Centro de Investigación para el Desarrollo Sustentable de Atacama (CRIDESAT), Universidad de Atacama. Copiapó, Chile.
Correspondig author: roberto.contreras@uda.cl
Fecha de Recepción: 23 agosto, 2017.
Fecha de Aceptación: 05 junio, 2018.
DOI:
2
IDESIA (Chile) Volumen Nº 2018
Sarcocornia individuals in the American continent,
recognizing four new species and confirming one of
those established by Scott (1978), named S. neei, S.
ambigua, S. andina, S. magellanica and S. pulvinata.
Sarcocornia is described as a pioneer plant
in marine environments, and is characterized
by extreme salt tolerance (Davy et al., 2006). In
the coastal Pacific, halophilic communities of
Sarcocornia grow in saline coastal marshes of Chile,
coastal regions of the south Pacific slope of Peru
(San Martín et al., 2006; Montesinos-Tubée, 2012),
and saltmarshes of the Andes of northern Chile
(Faúndez and Macaya, 1997). Due to their high salt
tolerance, both Salicornia and Sarcocornia possess
significant potential as a production model for arid
and saline environments (Katschnig et al., 2013);
they have by high nutritional value and high biomass
yield in field conditions (Ventura and Sagi, 2013).
Salicornia is presently cultivated commercially for
human consumption in Israel; it has been introduced
to the European market as a vegetable with leafless
shoots resembling green asparagus, and is in great
demand in gourmet kitchens due to its high mineral
and antioxidant content (Ventura et al., 2011; Lu et al.,
2010). In aquaculture production, a diet of Salicornia
bigelovii flour supplied to juveniles of the blue shrimp
L. stylirostris in intensive production systems has
produced positive results in terms of growth and
survival, and offers a low-cost alternative to more
expensive fish- and corn-based flour (Acosta-Ruiz
et al., 2011).
Salicornia and Sarcocornia show marked
morphological similarity; they are distinguished
phenotypically by inflorescence characteristics and
life form (Steffen et al., 2015). However, according to
studies on the basis of morphological characteristics
by Judd and Ferguson (1999) including flower
arrangement and growth habit, these features alone
are insufficient to distinguish the genera confidently.
Molecular markers could serve as a modern approach
for identification of these plants.
Many types of molecular techniques have been
used to identify plants of a wide range of varieties,
species and genera, including RFLP, AFLP, RAPD
and ASP-PCR (allele-specific primer PCR). Among
these, allele-specific primer PCR by agarose gel
electrophoresis is recognized as an efficient approach
for cultivar identification (Soleimani et al., 2003).
Likewise, diagnostic PCR using specific primers
offers a cost-effective alternative for molecular
identification of specific plant taxa. SNPs (single
nucleotide polymorphisms) can be detected using
allele-specific PCR primers designed in such a
manner that the 3’ nucleotide of a primer corresponds
to the site of the SNP (Ugozzoli and Wallace, 1991).
This technique allows preferential amplification of
one allele relative to another on account of the primers
being complementary to the site of DNA (Ugozzoli &
Wallace 1991). Using this technique, SNPs from ETS
sequences of Salicornia and Sarcocornia (Steffen et
al., 2015) could potentially be applied to discriminate
between genera.
The goal of this study was to design allele-specific
primers for amplification of short fragments from
ETS sequences of Sarcocornia and Salicornia, and to
evaluate an efficient diagnostic PCR to discriminate
Salicornia and Sarcocornia using samples from the
Atacama, Coquimbo and Los Lagos regions of Chile
and Salicornia control samples.
Materials and Methods
Plant Materials
Eighty two Sarcocornia neii plants from the
Atacama Region and seven S. neii plants from the
Coquimbo Region were sampled during 2016. Plants
were selected randomly and located by GPS as shown
in Table 1. Two DNA samples of Salicornia europaea,
supplied by Dr. Dirk Albach (Institut für Biologie
und Umweltwissenschaften, Carl von OssietzkyUniversität, Germany), were used as positive
Salicornia controls. Salicornia 1 control (ecotype 1)
and Salicornia 2 control (ecotype 2) were collected
by Dr. Albach near mudflats and low salt marshes
in Spiekeroog Island (Germany), respectively. Two
DNA samples of Sarcocornia perennis from Spain
and two DNA samples of Sarcocornia neii from
Puerto Mont (41º25’19.31’’S 72º53’42’’W, Los
Lagos region) were used as a positive Sarcocornia
control. Sarcocornia plants used as DNA controls
were identified morphologically according to Scott’s
approach. All samples were processed and analyzed
in the CRIDESAT Research Center, University of
Atacama.
Plant DNA Extraction
Genomic DNA from eighty nine plants was
extracted by a modified CTAB method following the
procedure described by Doyle and Doyle (1987). Five
grams of stem material were placed in sterile mortars
Rapid diagnostic PCR method for identification of the genera Sarcocornia and Salicornia
3
Table 1: Origin, location and samples collected in the Atacama and Coquimbo regions of Chile.
Samples
Latitude (ºS)
Longitude (ºW)
Altitude (m)
Samples
Latitude (ºS)
Longitude (ºW)
Altitude (m)
RCOP02
27°19’22.9”S
70°50’39.2”W
60
CLTO54
27°49’49.2”S
71°05’12.3”W
4
RCOP03
27°19’22.8”S
70°50’38.9”W
61
CLTO55
27°49’49.0”S
71°05’12.4”W
4
RCOP04
27°19’23.0”S
70°50’39.3”W
56
CLTO56
27°49’48.7”S
71°05’11.6”W
3
RCOP05
27°19’23.3”S
70°50’39.8”W
58
CLTO57
27°49’48.7”S
71°05’11.1”W
2
PVCO08
27°12’31.1”S
70°57’05.9”W
1
CLTO58
27°49’48.4”S
71°05’10.9”W
4
PVCO09
27°12’31.6”S
70°57’05.3”W
1
CLTO59
27°49’48.2”S
71°05’10.4”W
2
PVCO10
27°12’32.5”S
70°57’03.5”W
1
CLTO60
27°49’48.4”S
71°05’10.2”W
1
PVCO11
27°12’33.4”S
70°57’02.2”W
1
CLTO61
27°49’47.5”S
71°05’09.8”W
3
PVCO12
27°12’35.3”S
70°57’01.3”W
1
CLTO62
27°49’47.4”S
71°05’09.6”W
2
DECO13
27°16’39.6”S
70°56’32.6”W
0
CLTO63
27°49’54.6”S
71°05’11.5”W
7
DECO14
27°16’39.9”S
70°56’32.4”W
0
CLTO64
27°49’53.8”S
71°05’11.8”W
4
DECO15
27°16’40.1”S
70°56’32.2”W
0
CABJ65
28°05’16.1”S
71°08’27.3”W
6
DECO16
27°16’40.3”S
70°56’32.0”W
0
CABJ66
28°05’15.9”S
71°08’27.7”W
4
DECO17
27°16’40.6”S
70°56’31.6”W
0
CABJ67
28°05’16.0”S
71°08’27.9”W
3
DECO18
27°16’41.1”S
70°56’31.1”W
0
CABJ68
28°05’16.4”S
71°08’28.4”W
4
DECO19
27°16’41.5”S
70°56’30.9”W
0
CABJ69
28°05’16.7”S
71°08’29.8”W
5
DECO20
27°16’42.1”S
70°56’30.7”W
0
CABJ70
28°05’16.7”S
71°08’30.4”W
5
DECO21
27°16’42.7”S
70°56’30.2”W
0
CABJ71
28°05’16.7”S
71°08’31.9”W
3
DECO22
27°16’43.4”S
70°56’29.0”W
0
CABJ72
28°05’15.9”S
71°08’32.8”W
3
DECO23
27°16’57.4”S
70°56’16.4”W
0
CABJ73
28°05’15.2”S
71°08’32.8”W
5
DECO24
27°16’57.4”S
70°56’16.5”W
0
CABJ74
28°05’14.2”S
71°08’32.8”W
7
DECO25
27°17’22.6”S
70°56’02.8”W
0
CABJ75
28°05’13.4”S
71°08’33.4”W
5
DECO26
27°17’22.7”S
70°56’02.9”W
0
CABJ76
28°05’13.3”S
71°08’33.6”W
2
DECO27
27°17’29.0”S
70°56’00.0”W
0
CABJ77
28°05’12.4”S
71°08’33.4”W
4
DECO28
27°17’29.2”S
70°56’00.0”W
0
CABJ78
28°05’12.1”S
71°08’33.7”W
4
DECO29
27°17’38.0”S
70°55’56.2”W
0
CABJ79
28°05’11.3”S
71°08’34.0”W
3
DECO30
27°17’38.2”S
70°55’56.2”W
0
PBTO81
27°54’01.8”S
70°57’05.3”W
151
DECO31
27°17’55.2”S
70°55’48.8”W
0
PBTO82
27°54’01.7”S
70°57’05.2”W
151
DECO32
27°17’55.3”S
70°55’49.0”W
0
PBTO83
27°54’01.4”S
70°56’55.6”W
154
DECO33
27°18’12.5”S
70°55’43.8”W
0
PBTO84
27°54’01.8”S
70°56’55.4”W
154
DECO34
27°18’12.7”S
70°55’43.8”W
0
PBTO85
27°54’02.1”S
70°56’54.8”W
153
DECO37
27°19’03.4”S
70°55’06.8”W
4
PBTO86
27°54’02.2”S
70°56’54.5”W
152
DECO38
27°19’03.5”S
70°55’07.0”W
4
PBTO87
27°54’02.2”S
70°56’54.6”W
152
DECO39
27°19’03.7”S
70°55’07.1”W
5
VAHS88
28°27’55.0”S
71°12’23.8”W
8
DECO43
27°19’15.8”S
70°55’04.0”W
8
VAHS89
28°27’54.9”S
71°12’23.8”W
8
DECO44
27°19’15.7”S
70°55’04.0”W
7
VAHS90
28°27’54.8”S
71°12’23.4”W
9
DECO45
27°19’16.0”S
70°55’04.1”W
6
VAHS91
28°27’54.9”S
71°12’23.0”W
10
CLTO46
27°49’50.5”S
71°05’15.1”W
2
VAHS92
28°27’55.1”S
71°12’22.9”W
10
CLTO47
27°49’50.5”S
71°05’14.8”W
1
LASE93
29°57’40.4”S
71°19’19.4”W
3
CLTO48
27°49’50.5”S
71°05’14.4”W
1
LASE94
29°57’40.3”S
71°19’18.8”W
3
CLTO49
27°49’50.3”S
71°05’14.0”W
2
LASE95
29°57’40.5”S
71°19’21.7”W
4
CLTO50
27°49’50.3”S
71°05’13.6”W
3
LASE96
29°57’40.3”S
71°19’22.9”W
4
CLTO51
27°49’50.3”S
71°05’13.2”W
2
LASE97
29°57’39.9”S
71°19’11.2”W
4
CLTO52
27°49’49.8”S
71°05’12.6”W
2
LASE98
29°57’41.1”S
71°19’13.1”W
6
CLTO53
27°49’49.4”S
71°05’12.5”W
3
LASE99
29°57’40.9”S
71°19’14.6”W
7
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IDESIA (Chile) Volumen Nº 2018
at -80 ºC for a period of 12 h, and a fine powder
produced. About 500 mg of powder was placed in
2 mL tubes and mixed with 850 µL of preheated
(65 ºC) extraction buffer [100 mM Tris-HCl pH
8.0, 1.4 M NaCl, 20 mM EDTA, 2% (w/v) CTAB],
supplemented with 0.7% β-mercaptoethanol and
3% PVP-40. Samples were vortexed vigorously 10
s and incubated 1 h at 65 ºC. Mixes were inverted
three times during the incubation. The aqueous
phase was recovered by centrifuging at 14000 rpm
for 10 min. The recovered volume was mixed with
an equal volume of chloroform-isoamyl alcohol
(24:1). The mix was inverted gently over a period
of 2 min. Centrifugation at 14000 rpm for 10 min
was repeated. The upper phase was transferred to
a new tube and treated with 5 µL RNase A (100
µg/mL) at 37 ºC for 30 min. The extraction was
mixed with two-thirds volume isopropanol at
-20 ºC. The mix was inverted gently thirty times
and incubated on ice for 40 min. Centrifugation
at 14000 r pm for 10 min was repeated. The
supernatant was discarded and a DNA pellet was
obtained, to which a washing solution of 600 µL
of 70% ethanol and 10 mM ammonium acetate
was added; this was followed by centrifuging for 2
min at 14000 rpm. The wash was repeated twice.
The washing solution was discarded and the DNA
dried for 15 min at RT. To elute DNA, 20 µL TE
was added and incubated overnight at 4 °C. Quality
and concentration of total DNA were verified by
Colibri Microvolume Spectrophotometer (Titertek
Berthold, Germany) at 260, 280 and 230 nm, and
genomic DNA integrity checked on 0.7% agarose
gels.
Specific primer design
Specific primers were designed using ETS
(external transcribed spacer) sequences of ribosomal
RNA from nine Salicornia species and eight
Sarcocornia species previously obtained by Kadereit
et al. (2007). The accession numbers of these 17 ETS
sequences were obtained from GenBank (Figure 1).
The sequences were aligned by MEGA 7.0 software
and ClustalW as shown in Figure 1. Oligo® software
was used to design three specific primer pairs each for
Salicornia and Sarcocornia (Table 2) from conserved
SNP. These six potentially genus-specific primer
pairs were examined for their presence in other plant
species available in GenBank (BlastN option, http://
www.ncbi.nlm.nih.gov/).
PCR optimization
DNA control samples were used as a template
to investigate analytical sensitivity. Serial DNA
concentrations were prepared for each PCR reaction
at 1 ng, 5 ng, 10 ng, 20 ng and 30 ng to establish the
sensitivity limit of the PCR assay. PCR reactions
were performed in 12 µL final volume containing
(1ng to 30 ng) DNA, 0.85 µL each primer (5 µM)
and 6 µL DreamTaq PCR Master Mix 2X (Thermo
Scientific). Amplifications were carried out in Swift
Max Pro (Esco) and MultiGene Optimax (Labnet)
thermal cyclers with the following program: an initial
step of 4 min at 94 °C, 35 cycles of 30 s at 94 °C, 30
s at 54 °C and 2 min at 72 °C, followed by a final
extension step of 7 min at 72 °C. To determine the
optimum annealing temperature (Ta), a gradient
PCR was performed using DNA control samples
from the Sarcocornia control (from Puerto Montt),
and Salicornia 1 and 2 controls. The PCR mix and
program were identical to those indicated above, but
with optimized DNA concentration and a different
Ta. The Ta were 50 °C, 50.7 °C, 51.5 °C, 54 °C, 57.2
°C, 59.2 °C and 60 °C in order to obtain the optimum
temperature at which only the template DNA of
Sarcocornia or Salicornia would be amplified. All
PCR products were visualized on 1.5% TBE agarose
gels.
Multiplex PCR and allele-specific PCR
To demonstrate that diagnostic PCR works
well with specific Sarcocornia and Salicornia
DNA primer pairs, a primer pair from plastid trnL
(UAA) intron (~110 pb) was used as a positive plant
control in the same PCR reaction. The multiplex
PCR to amplify plastid trnL (UAA) intron and
the ETS region from Salicornia and Sarcocornia
were performed under conditions similar to those
used for PCR optimization, except for the primer
concentrations: 1 µL of each primer SALI or
SARCO to 5 µM and 0.7 µL of each trnL primer to
5 µM (F: 5’GGGCAATCCTGAGCCAA 3’ R: 5’
CCATTGAGTCTCTGCACCTATC 3’; Taberlet
et al., 2006). The amplification of allele-specific
diagnostic PCR was performed using 3 primers
simultaneously in a reaction tube with genomic DNA
from Sarcocornia and Salicornia controls. Diagnostic
PCR reactions were performed in 16 µL final volume
containing 20 ng DNA, 0.55 µL SARCO3F primer,
1.3 µL SARCO1R, 0.55 µL SARCO3R (all primers
Rapid diagnostic PCR method for identification of the genera Sarcocornia and Salicornia
5
Figure 1: Position of specific primers in the ETS (external transcribed spacer) sequences of ribosomal RNA of nine Salicornia and
eight Sarcocornia available in GenBank. Forward primer was colored in light grey and reverse primer in dark grey. Primers with
SNP in position 3’ were shown in white letters.
6
IDESIA (Chile) Volumen Nº 2018
Table 2: Sequences, temperature and SNP information of the primers designed.
Primer name
Sequence (5’-3’)
SALI1F
GATGCGGTACGTGATGGT
SALI1R
CCACACGTCGCCCAAGG
SALI2F
TCTTTGCTTGTGCATTGG
SALI2R
CGGACGTAGAGCGAATA
SALI3F
ATGCTGCAAGTGCACCATTTT
SALI4R
TCATGCTTGTTTTCACAAA
SARCO1F
CTCTATGCTTGTGCATTGA
SARCO1R
GTGCTTGTTTTCGCTTG
SARCO2F
TGATGCGGTACGTGTTGGC
SARCO2R
AACAGTCCGCTCGACCTCC
SARCO3F
CCCTATGTTGGATTCCTATTG
SARCO3R
CATCCATCATCAGCGTAC (*)
Target taxa
Salicornia sp.
Salicornia sp.
Salicornia sp.
Sarcocornia sp.
Sarcocornia sp.
Sarcocornia sp.
Tm °C
54.3
51.9
52.3
52.5
55.9
52.3
Length of base pair
SNP
18
T/C
17
--
18
G/A
17
--
21
T/G;T/-
19
T/C;T/A
19
A/G
17
C/T;A/T
19
C/T
19
--
21
G/A
18
--
Length of PCR product
138
155
176
221
232
243
(*) sequence not shown in Figure 1
at 5 µM), and 8 µL DreamTaq PCR Master Mix 2X
(Thermo Scientific). Amplifications were carried out
in Swift Max Pro (Esco) and MultiGene Optimax
(Labnet) thermal cyclers, with the following program:
an initial step of 4 min at 94 °C, 35 cycles of 30 s at
94 °C, 30 s at 52 °C and 2 min at 72 °C, followed
by a final extension step of 7 min at 72 °C. All PCR
assays were repeated three times and PCR products
visualized on 1.5% TBE agarose gels.
Results and Discussion
Various genetic techniques have been used to
authenticate differences between different plant
cultivars, varieties, species and genera. Recently
the application of diverse molecular approaches has
allowed the validation of plant species that previously,
due to the difficulty in identifying plants based on
morphological characteristics, were considered to
be of questionable taxonomic status (Nybom et al.,
2014). Systematic establishment of phylogenetic
relationships in Salicornia and Sarcocornia using
morphological markers is difficult due to the lack
of or significant reduction of principal structures
such as leaves and flowers (Kadereit et al., 2006).
Morphological characteristics such as perennial
habitat and similarity in flower size were the main
basis of the argument to distinguish Sarcocornia from
Salicornia (Scott, 1978). However, there are species
such as Sarcocornia natalensis and Sarcocornia
freitagii containing short-lived and herbaceous
perennials. Therefore, the traditional characteristics
used to identify these genera are currently insufficient
(Steffen et al., 2015).
In this study, six primer pairs were designed to
evaluate allele-specific amplifications from Salicornia
and Sarcocornia. For this, the 3’ end base of the forward
or reverse primer was positioned strictly on the SNP.
Prior to plant identification, two PCR optimization tests
were performed using control DNA from each genus.
The results of testing for PCR sensitivity showed good
amplification of the expected fragments using samples
containing between 1 and 20 ng per DNA/PCR reaction
(Figure 2). Therefore, to ensure the effectiveness of the
majority of PCR assays it was decided to employ a
final volume of 15 ng of DNA/14 µL PCR.
To evaluate the effect of annealing temperature
(Ta), Salicornia and Sarcocornia control DNA was
amplified using six primer pairs, using a Ta between
50 °C and 60 °C. The results of the diagnostic PCR
based on the SALI1F-SALI1R primer pair indicated
the presence of a well-defined fragment of expected
size and high intensity in Salicornia controls 1 and 2,
whereas the same robust fragment was observed in
the Sarcocornia control at all annealing temperatures
(Figure 3). In contrast, correct amplifications were
observed in both Salicornia controls with the
SALI2F-SALI2R primer pair at almost all annealing
temperatures, while the Sarcocornia control showed
no amplification of fragments at different annealing
temperatures. Secondary PCR products with
SALI2F-SALI2R were observed in the Salicornia 1
control, however, these PCR products disappeared
at Ta greater than 57.2 °C. The Salicornia controls
of the SALI3F-SALI4R primer pair resulted in
good amplification of fragments at all annealing
temperatures, but not in the Sarcocornia control,
which showed no amplification (Figure 3).
Rapid diagnostic PCR method for identification of the genera Sarcocornia and Salicornia
7
Figure 2: Agarose gel electrophoresis of PCR products obtained from the sensitivity test (1 ng to 30 ng) of primer pair SARCO1FSARCO1R and SALI3F-SALI4R. Lanes 1, 3, 5, 7 and 9 = Sarcocornia control; 2, 4, 6, 8 and 10 = DECO19; 11, 13, 15, 17 and
19 = Salicornia 2 control; and 12, 14, 16, 18 and 20 = Salicornia 1 control. MP: 100 bp DNA ladder.
Figure 3: Electrophoresis of DNA amplified fragments obtained from annealing temperature (Ta) tests with Sarcocornia control (Sarcocornia Puerto Montt), Salicornia 1 control and Salicornia 2 control, using primer pair SALI1F-SALI1R (138 bp),
SALI2F-SALI2R (155 bp), SALI3F-SALI4R (176 bp), SARCO1F-SARCO1R (221 bp), SARCO2F-SARCO2R (232 bp) and
SARCO3F-SARCO3R (243 bp). MP: 100 bp DNA ladder.
8
IDESIA (Chile) Volumen Nº 2018
PCR based on the SARCO1-SARCO1F primer
pair indicated the presence of a fragment of expected
size in the Sarcocornia control, but no fragment was
observed at any annealing temperature for Salicornia
controls 1 and 2 (Figure 3). Amplification of fragments
in both Salicornia and Sarcocornia controls was
detected at different annealing temperatures using the
SARCO2F-SARCO2R primer pair. Unlike previous
primer pairs, the SARCO3F-SARCO3R primer pair
showed good amplification of PCR fragments based
on the Sarcocornia control at different annealing
temperatures, although the Salicornia controls also
showed fragment amplification at a temperature of 54
°C; amplification was not observed at temperatures
over 57.2 °C (Figure 3).
Finally, two primer pairs (SALI1F-SALI1R
and SARCO2F-SARCO2R) were discarded
since they produced amplification in controls of
both Sarcocornia and Salicornia. Each of these
primer pairs had one SNP in the 3’ end position of
the forward primer. However, in the SARCO2FSARCO2R primer pair differences in amplification
between Sarcocornia and Salicornia controls were
observed at annealing temperatures of up to 68 °C,
whereas no amplification differences were observed
with the SALI1F -SALI1R primer pair at annealing
temperatures of up to 72 °C (data not shown). The
reason for this is that in most cases a single base pair
change at the 3’ end is not a sufficient basis for reliable
discrimination (Kwok et al., 1994); in fact, this is the
main reason that allele-specific primer techniques are
not widely used. Otherwise, despite having one SNP
in the 3’ position on the forward primer, SALI2FSALI2R and SARCO3F-SARCO3R showed a good
match in controls of the genera. However, large
genus differences were demonstrated efficiently
with SARCO1F-SARCO1R and SALI3F-SALI4R
compared to other primer pairs. These primer pairs
performed optimally because three consecutive SNPs
from the 3’ position were considered in the process
of primer design. The high specificity of primer pairs
SARCO1F-SARCO1R and SALI3F-SALI4R for
genus detection is due to an increase in the number
of SNP targets which were designed, such as forward
and reverse primers.
To evaluate PCR amplification with a positive
control fragment (trnL intron), primers selected to
identify Sarcocornia and Salicornia were tested in a
multiplex PCR assay including the trnL primer pair
in the same reaction. The results indicated that the
SARCO1F-SARCO1R and SARCO3F-SARCO3R
primer pairs produced amplification of PCR products
in Sarcocornia samples, whereas no amplification
was observed in Salicornia controls. Likewise, the
SALI2F-SALI2R and SALI3F-SALI4R primer pairs
resulted in amplification in the Salicornia controls,
but not in the Sarcocornia control. The trnL positive
control primer pair was observed in all DNA samples
mentioned above in a single band at ~110 pb (Figure
4A).
In another test, the diagnostic PCR by allelespecific primers (SARCO3F, SARCO1R and
Figure 4: Example of diagnostic PCR with Salicornia and Sarcocornia. A) Electrophoresis of DNA amplified fragments obtained
from Salicornia 1 control, Salicornia 2 control, Sarcocornia control (Puerto Montt), CABJ66 and DECO18 by means of multiplex
PCR with a primer set of the trnL region and primer pairs SARCO1F-SARCO1R (Ta=54°C), SARCO3F-SARCO3R (Ta=57°C),
SALI2F-SALI2R (54°C) and SALI3F-SALI4R (Ta=54°C). B) Agarose gel electrophoresis of amplified fragments with two replicates from Sarcocornia controls (1, 2 = Sarcocornia of Puerto Montt; 3, 4 = Sarcocornia of Spain) and Salicornia controls (5,
6 = Salicornia 2 control; 7, 8 = Salicornia 1 control) and with three primers used for allele-specific PCR (SARCO3F, SARCO1R
and SARCO3F). MP1: 100 bp DNA ladder and MP2: 1 kb DNA ladder.
Rapid diagnostic PCR method for identification of the genera Sarcocornia and Salicornia
SARCO3F) were used to amplify Sarcocornia
and Salicornia DNA in a simultaneous reaction.
The result confirmed the presence of one DNA
banding pattern for Sarcocornia and one DNA
banding pattern for Salicornia: the former pattern
with bands of 243 and 135 bp and the latter with
only one band of 243 bp (Figure 4B). However,
diagnostic PCR based on Spanish Sarcocornia
DNA produced a weak 234 bp band and a strong
135 bp band. The DNA of Sarcocornia showed
a band of 135 bp, while this was not observed in
Salicornia. In brief, using these specific primer
pairs in a PCR reaction followed by agarose
gel visualization offers a reliable method for
identification of Sarcocornia and Salicornia;
additionally, the detection and validation of SNPs
from ETS sequences of these genera, described by
Kadereit et al. 2007, was confirmed.
Using primer pairs appropriate for the
identification of both genera, we tested 82 samples
from the Atacama region, seven samples from the
Coquimbo region, and two samples each from
the Los Lagos region and from Spain. The results
showed positive amplification in all plants (82)
from the Atacama region using the SARCO1F1R and SARCO3F-3R primer pairs, however two
DNA samples (DECO23 and DECO32) did not
show amplification, and one sample (DECO21)
showed two PCR fragments using the SARCO3F-3R
primer pair (Table 3). The DNA samples from the
Coquimbo region, the Los Lagos region and Spain
had positive amplification using the SARCO1F-1R
and SARCO3F-3R primer pairs (Table 3). None of the
DNA samples from any of the regions demonstrated
amplification with the control primer SALI 2F-2R
and SALI 3F-4R, although only the CABJ74 sample
showed one PCR fragment similar to the expected
size with SALI2F-2R. The results of the present
study are consistent with the report of Alonso and
Crespo (2008), indicating the predominance of
Sarcocornia in Chile; this suggests that the approach
is efficiently designed for the detection of plants of
this genus. In addition, we believe that the method
could theoretically be applied in other parts of the
world, since the primer pairs were designed from
9
representative Sarcocornia ETS sequences of three
continents.
In a further step, this PCR diagnostic method
was applied using Sarcocornia tissue from
commercial products in which clear identification
of genus (data not shown) was possible. This
discrimination method could therefore potentially
be useful for market quality control purposes,
particularly given that distinctions on the basis of
morphological characteristics (flower) and growth
habit (area) are obviously not feasible in the case
of final packaged products (sea asparagus). The
potential for a molecular approach in this context
is of particular interest considering the differences
in nutritional and agronomic characteristics of
the genera; Sarcocornia has a saltier taste than
Salicornia, verified by an elevated EC (electrical
conductivity) value in shoots immersed in seawater
(Ventura et al., 2011), and although both possess
high total lipid content relative to other plants
using culture in seawater, the Salicornia ecotype
exceeds Sarcocornia in terms of total fatty acids
and omega-3 percentage. Furthermore, the annual
Salicornia ecotype has higher yields than perennial
Sarcocornia ecotypes (Ventura et al., 2011). In
summary, a PCR-based genotyping method has
been developed to discriminate between Salicornia
and Sarcocornia using only DNA, PCR and
electrophoresis.
Conclusion
In this study, a simple and reliable method
through four specific-primers (SALI2F-2R, SALI3F4R, SARCO1F-1R and SARCO3F-3R), multiplex
PCR and allele specific-primers has been developed
to discriminate between Salicornia and Sarcocornia.
Acknowledgments
We sincerely thank Prof. César Benito
(Departamento de Genética, Universidad Complutense
de Madrid) for comments that greatly improved the
paper and Niklas Buhk for assistance with Salicornia
samples.
Primer pair
10
Table 3: Summary of the results of positive (+) and negative (-) PCR amplifications with different primer pairs on each sample.
Primer pair
SARCO 3F
SARCO 3R
(Ta=57.2°C)
SALI 2F
SALI 2R
(Ta=54°C)
SALI 3F
SALI 4R
(Ta=54°C)
-
CLTO55
+
+
-
-
-
CLTO56
+
+
-
-
-
-
CLTO57
+
+
-
-
-
-
CLTO58
+
+
-
-
+
-
-
CLTO59
+
+
-
-
+
+
-
-
CLTO60
+
+
-
-
PVCO10
+
+
-
-
CLTO61
+
+
-
-
PVCO11
+
+
-
-
CLTO62
+
+
-
-
PVCO12
+
+
-
-
CLTO63
+
+
-
-
DECO13
+
+
-
-
CLTO64
+
+
-
-
DECO14
+
+
-
-
CABJ65
+
+
-
-
DECO15
+
+
-
-
CABJ66
+
+
-
-
DECO16
+
+
-
-
CABJ67
+
+
-
-
DECO17
+
+
-
-
CABJ68
+
+
-
-
DECO18
+
+
-
-
CABJ69
+
+
-
-
DECO19
+
+
-
-
CABJ70
+
+
-
-
DECO20
+
+
-
-
CABJ71
+
+
-
-
DECO21
+
+(*)
-
-
CABJ72
+
+
-
-
DECO22
+
+
-
-
CABJ73
+
+
-
-
DECO23
+
-
-
-
CABJ74
+
+
+(**)
-
DECO24
+
+
-
-
CABJ75
+
+
-
-
DECO25
+
+
-
-
CABJ76
+
+
-
-
DECO26
+
+
-
-
CABJ77
+
+
-
-
DECO27
+
+
-
-
CABJ78
+
+
-
-
DECO28
+
+
-
-
CABJ79
+
+
-
-
DECO29
+
+
-
-
PBTO81
+
+
-
-
DECO30
+
+
-
-
PBTO82
+
+
-
-
DECO31
+
+
-
-
PBTO83
+
+
-
-
Sample
SARCO 3F
SARCO 3R
(Ta=57.2°C)
SALI 2F
SALI 2R
(Ta=54°C)
SALI 3F
SALI 4R
(Ta=54°C)
RCOP02
+
+
-
RCOP03
+
+
-
RCOP04
+
+
RCOP05
+
+
PVCO08
+
PVCO09
IDESIA (Chile) Volumen Nº 2018
Sample
SARCO 1F
SARCO 1R
(Ta=54°C)
SARCO 1F
SARCO 1R
(Ta=54°C)
Continuación Table 3:
Primer pair
Primer pair
SARCO 3F
SARCO 3R
(Ta=57.2°C)
SALI 2F
SALI 2R
(Ta=54°C)
SALI 3F
SALI 4R
(Ta=54°C)
-
PBTO84
+
+
-
-
-
-
PBTO85
+
+
-
-
-
-
PBTO86
+
+
-
-
+
-
-
PBTO87
+
+
-
-
+
+
-
-
VAHS88
+
+
-
-
DECO39
+
+
-
-
VAHS89
+
+
-
-
DECO43
+
+
-
-
VAHS90
+
+
-
-
DECO44
+
+
-
-
VAHS91
+
+
-
-
DECO45
+
+
-
-
VAHS92
+
+
-
-
CLTO47
+
+
-
-
LASE93
+
+
-
-
CLTO48
+
+
-
-
LASE94
+
+
-
-
CLTO49
+
+
-
-
LASE95
+
+
-
-
CLTO50
+
+
-
-
LASE96
+
+
-
-
CLTO51
+
+
-
-
LASE97
+
+
-
-
CLTO52
+
+
-
-
LASE98
+
+
-
-
CLTO53
+
+
-
-
LASE99
+
+
-
-
-
Sample
SARCO 3F
SARCO 3R
(Ta=57.2°C)
SALI 2F
SALI 2R
(Ta=54°C)
SALI 3F
SALI 4R
(Ta=54°C)
DECO32
+
-
-
DECO33
+
+
DECO34
+
+
DECO37
+
DECO38
CLTO54
+
+
-
-
Spain Sarcocornia1
+
+
-
-
Sarcocornia Puerto Mo.
+
+
-
Spain Sarcocornia2
+
+
-
-
Sarcocornia Puerto Mo.
+
+
-
-
Salicornia 1 control
-
-
+
+
Salicornia 2 control
-
-
+
+
(*) two fragments amplified
Rapid diagnostic PCR method for identification of the genera Sarcocornia and Salicornia
Sample
SARCO 1F
SARCO 1R
(Ta=54°C)
SARCO 1F
SARCO 1R
(Ta=54°C)
(**) one fragment amplified similar to expected size.
11
12
IDESIA (Chile) Volumen Nº 2018
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