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Journal of the Science of Food and Agriculture
J Sci Food Agric 80:29±32 (2000)
Genetic differentiation between sole (Solea
solea) and Greenland halibut (Reinhardtius
hippoglossoides) by PCR–RFLP analysis of a
12S rRNA gene fragment
Ana Céspedes,* Teresa Garcı́a, Esther Carrera, Isabel González, Alicia Fernández,
Luis Asensio, Pablo E Hernández and Rosario Martı́n
Departamento de Nutrición y Bromatologı́a III, Facultad de Veterinaria, Universidad Complutense, E-28040 Madrid, Spain
Abstract: PCR±RFLP analysis was applied to the identi®cation of two closely related ¯at®sh species:
sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Ampli®cation of DNA
isolated from ®sh muscle samples was carried out using a set of primers ¯anking a region of 321 base
pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on
sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI
endonucleases. The restriction pro®les obtained by agarose gel electrophoresis when amplicons were
cut with AciI and MwoI enzymes allowed the unequivocal identi®cation of sole and Greenland halibut
species.
# 2000 Society of Chemical Industry
Keywords: species identi®cation; ¯at®sh; PCR±RFLP; 12S rRNA; Solea solea; Reinhardtius hippoglossoides
INTRODUCTION
Substitution of cheaper for more expensive ®sh species
is dif®cult to detect when the usual external characteristics of the ®sh have been removed.1 Greenland
halibut (Reinhardtius hippoglossoides) ®llets are often
labelled and marketed as sole (Solea solea) owing to the
greater popularity of and higher consumer demand for
this latter species.2,3 Non-morphological methods are
then necessary for the authentication of ®sh species
and the enforcement of labelling regulations.
Various approaches have been used to address this
problem, including electrophoretic, chromatographic,
immunological and genetic techniques.4,5 Methods
based on DNA analysis have two major advantages:5,6
(i) DNA is an extremely stable and long-lived
biological molecule; (ii) the information it holds is
richer than that in any other biomolecule.
The development of molecular techniques that
identify nucleotide-level DNA sequence polymorphisms between species has created an unlimited source
of genetic markers which can be used for species
identi®cation.7,8 Several nuclear DNA (nDNA)
markers have been described for ®sh including the
preprogonadotropin-releasing hormone (GnRH)
gene,9 the alpha-actin gene,10 the growth hormone
gene11 and the nuclear 5S ribosomal RNA (rRNA)
gene.12 Nevertheless, the design of a great variety of
universal primers for polymerase chain reaction (PCR)
ampli®cation of speci®c mitochondrial DNA
(mtDNA) sequences13±16 has promoted the use of
mtDNA markers for ®sh species identi®cation.1,17±21
Once the targeted DNA fragments have been
ampli®ed, they may be sequenced subsequently14 or
digested with restriction endonucleases and subjected
to restriction fragment length polymorphism analysis
(PCR±RFLP).15,17±22 PCR±RFLP is much simpler
and less costly than DNA sequencing and nucleotide
sequence analysis. This technique, combining simplicity, speed, resolving power and low cost, is an
interesting approach to ®sh species identi®cation for
large-scale studies such as required in inspection
programmes.
We describe in this paper a method for differentiation between sole (Solea solea) and Greenland halibut
(Reinhardtius hippoglossoides) based on PCR ampli®cation and restriction site polymorphism analysis of a
conserved region in the mitochondrial 12S rRNA
gene.
* Correspondence to: Ana Céspedes, Departamento de Nutrición y Bromatologı́a III (Higiene y Tecnologı́a de los Alimentos), Facultad de
Veterinaria, Universidad Complutense, E-28040 Madrid, Spain
E-mail: anaces@eucmax.sim.ucm.es
Contract/grant sponsor: Comisión Interministerial de Ciencia y Tecnologı́a, (CICYT), Spain; contract/grant number: ALI98-0696
Contract/grant sponsor: Comunidad Autónoma de Castilla–La Mancha
Contract/grant sponsor: Ministerio de Educación y Ciencia (Spain)
(Received 4 January 1999; revised version received 4 May 1999; accepted 8 July 1999)
# 2000 Society of Chemical Industry. J Sci Food Agric 0022±5142/2000/$17.50
29
A CeÂspedes et al
EXPERIMENTAL
Sample selection and DNA extraction
12S3: 5'- TAA CAT CCA CCT AGA
GGA GCC -3' (21mer)
Sole (Solea solea) samples were obtained from MercaMadrid (Madrid, Spain). Frozen samples of Greenland halibut (Reinhardtius hippoglossoides) were
acquired from ¯at®sh manufacturers in Vigo and
Valencia (Spain). All specimens were morphologically
identi®ed.23
Genomic DNA was extracted from ®sh muscle
samples according to a previously described procedure.20 Fifteen individuals of each species were
analysed.
Sequence analysis and primer design were performed with the aid of the Wisconsin Package, Version
9.0 (Genetics Computer Group, Madison, WI, USA)
and the Amplify software.25
DNA was ampli®ed following the protocol mentioned above for PCR ampli®cation, except that (a)
primer 12S3 was used instead of primer 12S1 and (b)
only 100 ng of DNA and 10 pm of each primer were
necessary. The primer annealing temperature was
55 °C.
PCR amplification of a 436bp fragment from the 12S
rRNA gene
Restriction site mapping and enzymatic digestion of
the 321bp 12S rRNA gene fragment
DNA was ampli®ed by PCR in 50 ml reaction volume
containing 10 mM Tris±HCl pH 8.8, 1.5 mM MgCl2,
50 mM KCl, 1 g lÿ1 Triton X-100, 0.2 mM each of
dATP, dTTP, dGTP and dCTP, 30 pmol of each
primer, 100±1500 ng of template DNA and 2 U of
DynaZyme II DNA polymerase (Finnzymes Oy,
Espoo, Finland). The set of primers used for ampli®cation consisted of 12S1 and 12S2 oligonucleotides:24
12S1: 5'- AAA CTA GGA TTA GAT
ACC CTA TTA T -3' (25mer)
12S2: 5'- AAG AGC GAC GGG
CGA TGT GT -3' (20mer)
PCR ampli®cation was performed in a Progene
Thermal Cycler (Techne Ltd, Cambridge, UK).
Thirty-®ve cycles of ampli®cation with the following
step cycle pro®le were carried out: strand denaturation
at 93 °C for 30 s, primer annealing at 50 °C for 30 s and
primer extension at 72 °C for 45 s. An initial denaturation at 93 °C for 2 min and a ®nal extension at 72 °C for
5 min improved the product yield. The ampli®cation
products were tested on 15 g lÿ1 agarose gel.
Sequencing of the 436bp PCR products
The DNA fragments ampli®ed with 12S1 and 12S2
oligonucleotides from sole and Greenland halibut
species were puri®ed using the Qiaquick gel extraction
kit (Qiagen GmbH, Hilden, Germany) according to
the manufacturer's instructions. Puri®ed PCR products were sequenced at the Centro de Investigaciones
BioloÂgicas (Consejo Superior de Investigaciones
Cientõ®cas, Madrid, Spain). DNA sequencing was
accomplished in an ABI Prism model 377 DNA
sequencer (Perkin-Elmer/Applied Biosystems Division, Foster City, CA, USA) using 12S1 and 12S2
primers and the dRhodamine terminator cycle sequencing ready reaction kit (Perkin-Elmer).
PCR amplification of a 321bp fragment from the 12S
rRNA gene
The 436 bp sequences from sole and Greenland
halibut 12S rRNA gene previously obtained were used
for the design of a new primer 12S3:
30
Restriction maps of 12S rRNA gene sequences were
obtained using the Wisconsin Package, Version 9.0.
AciI and MwoI endonucleases (New England BioLabs,
Beverly, MA, USA) were selected as suitable candidates for the identi®cation of sole and Greenland
halibut species.
PCR products ampli®ed using 12S3 and 12S2
primers were subjected to restriction digestion with
AciI and MwoI endonucleases, without previous puri®cation. All reactions were performed in 20 ml volumes
using digestion conditions speci®ed by the manufacturer.
DNA fragments were separated by electrophoresis
in 35 g lÿ1 MS8 (Hispanlab SA, Alcobendas, Spain)
agarose gels. Fragments were analysed using the
Geldoc 1000 UV Fluorescent Gel Documentation
System-PC (Bio-Rad Laboratories, Hercules, USA).
The molecular weight standard used to estimate fragment size was the 100 bp DNA ladder (GibcoBRL,
Life Technologies, Maryland, USA).
RESULTS AND DISCUSSION
Mitochondrial DNA has been widely used as a marker
in species identi®cation.22,26,27 Some distinguishing
traits of this molecule have made it feasible: (i) it has a
simple genetic structure, lacking complicated features
such as repetitive DNA, transposable elements,
pseudogenes and introns; (ii) it exhibits a straightforward mode of genetic transmission, without recombination or other genetic rearrangements; (iii)
mitochondrial genes in vertebrates have a high
mutation rate, making it possible to accumulate point
mutations quickly enough to allow, in most cases, the
discrimination of even closely related species; (iv)
mtDNA is easy to isolate.
The mitochondrial genome of animals contains 13
genes coding for proteins, 22 genes coding for transfer
RNA (tRNA), one major non-coding region and two
ribosomal RNA (rRNA) genes: the small 12S rRNA
gene (about 819±975 bp long in vertebrates) and the
large 16S rRNA gene (about 1571±1640 bp long in
vertebrates).26 Several factors have to be considered
before a speci®c segment of DNA is selected for PCR
J Sci Food Agric 80:29±32 (2000)
Sole and Greenland halibut identi®cation by PCR±RFLP
Figure 1. DNA sequences from part of the 12S rRNA gene of two sole
(Solea solea) and two Greenland halibut (Reinhardtius hippoglossoides)
samples, aligned with the homologous rainbow trout (Oncorhynchus
mykiss) sequence obtained from GenBank (Accession number L29771).
AciI and MwoI restriction sites are shown with shadow. Bold type
nucleotides indicate the position of primers 12S1, 12S2 and 12S3 used for
PCR amplification.
ampli®cation.20 The mitochondrial encoded gene for
12S rRNA satis®es most of them: it has an acceptable
length, an adequate grade of mutation and there are
various sequences available in the databases.
Mitochondrial primers 12S1 and 12S2 were
described by Simon et al 24 for PCR ampli®cation of
a conserved region from the 12S rRNA gene. These
oligonucleotides were designed to be universal in
insect identi®cation, but they have also been used
successfully in shrimp species.17 We employed 12S1
and 12S2 oligonucleotides in order to amplify a 436 bp
12S rRNA gene fragment useful for RFLP analysis.
However, although ampli®cation was achieved with
J Sci Food Agric 80:29±32 (2000)
DNA from both species, in the case of sole, amplicon
production was too low to allow a routine RFLP
analysis (data not shown).
The low yield of amplicon obtained from sole
samples could be due to the fact that the primers
used, or at least one of them, did not anneal properly
on the sole 12S rRNA sequence. For this reason, the
436 bp PCR fragments from two different individuals
of each sole and Greenland halibut sample were
sequenced. Comparison of the 12S rRNA gene
sequences obtained allowed the design of the 12S3
oligonucleotide (Fig 1). This primer, together with the
previously employed 12S2 oligonucleotide, improved
PCR ampli®cation, yielding a 321 bp fragment with a
high DNA concentration in both species analysed (Fig
2, lane 1).
The restriction pro®les of PCR products obtained
with 12S3 and 12S2 primers were analysed using the
MAP program in the Wisconsin Package. From the
detailed comparison of these restriction maps, we
hypothesised that AciI and MwoI endonucleases could
provide speci®c identi®cation of sole and Greenland
halibut. Restriction sites of these enzymes are marked
on the sequences (Fig 1).
Fig 2 shows the results obtained following restriction analysis of sole and Greenland halibut PCR
products after incubation with AciI and MwoI endonucleases. AciI (Fig 2, lanes 2 and 3) cleaved the
analysed 12S rRNA segment of sole into two
fragments of 249 and 72 bp, as expected from the
presence of only one recognition site, while the two
recognition sites present in Greenland halibut PCR
products yielded three fragments of 202, 72 and 47 bp.
Three restriction sites for MwoI were found in the
sequences of sole PCR products, yielding four DNA
fragments of 151, 101, 42 and 27 bp (Fig 2, lane 4),
whereas the two AciI restriction sites present in
Greenland halibut sequences yielded three DNA
fragments of 130, 128 and 63 bp (Fig 2, lane 5).
Fifteen individuals each of sole and Greenland
Figure 2. Electrophoretic analysis of the 12S rRNA gene PCR products
obtained with 12S3 and 12S2 oligonucleotides. Samples in lanes are: 1,
undigested PCR product; 2 and 4, sole (Solea solea); 3 and 5, Greenland
halibut (Reinhardtius hipoglossoides). Endonuclease AciI was used for
restriction digestion on samples 2 and 3. Endonuclease MwoI was used for
digestion on samples 4 and 5. M = 100 bp ladder for molecular weight
marker.
31
A CeÂspedes et al
halibut species were analysed using PCR±restriction
site analysis of the 12S rRNA gene. The results did not
show intraspeci®c polymorphism for the two restriction endonucleases tested.
PCR±RFLP of a conserved region in the 12S rRNA
gene allows the accurate identi®cation of sole and
Greenland halibut ¯at®sh species. Interpretation of
the restriction pro®les can be performed visually,
without the need for computer analysis.
ACKNOWLEDGEMENTS
This work was supported by grant ALI98-0696 from
the ComisioÂn Interministerial de Ciencia y TecnologõÂa
(CICYT), Spain. Ana CeÂspedes and Esther Carrera
are recipients of grants from the Comunidad
AutoÂnoma de Castilla±La Mancha and the Ministerio
de EducacioÂn y Ciencia (Spain) respectively. The
 ngel MendizaÂbal (SVO,
authors would like to thank A
MercaMadrid), Frigorõ®cos BerbeÂs SA (Vigo, Spain)
and Silomar (Valencia, Spain) for kindly supplying ®sh
samples.
12
13
14
15
16
17
18
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