Forensic Science International 151 (2005) 239247

www.elsevier.com/locate/forsciint

Development of a genetic traceability test in pig based on

single nucleotide polymorphism detection
F. Goffaux, B. China *, L. Dams, A. Clinquart, G. Daube
Department of Food Sciences, Faculty of Veterinary Medicine, University of Lie`ge,
Boulevard de Colonster, 20/B43bis, 4000 Liege, Belgium
Received 8 November 2004; received in revised form 22 February 2005; accepted 22 February 2005
Available online 19 April 2005

Abstract
In order to assure traceability along the meat transformation process, a powerful system is required. The administrative
traceability shows limits that the use of genetic markers could overcome. The individual genomes contain sequence differences,
basis of the genetic polymorphism of which the genetic markers are the witnesses. Among them, two classes seem to dominate
on the traceability field: the microsatellites and the single nucleotide polymorphisms (SNP). The aim of this work was to develop
a genetic traceability test in pig based on SNPs mainly located in 50 and 30 untranslated regions (UTRs). A set of 21 SNP markers
including new SNPs identified in this study and SNPs previously described was selected. A genotyping assay was performed on
96 individuals representing the major crossbred of the pig population in Belgium. Results showed that all individuals tested
presented a different genotype. This genotyping method might help the administrative system to guarantee the traceability of
pork meat along the transformation process.
# 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pig; Single nucleotide polymorphism; Traceability

1. Introduction
During the last years in Belgium, the food industry was
affected by several scandals and crisis such as the hormones,
the polychlorinated biphenyl (PCB) (unjustly called dioxin
crisis) and the bovine spongiform encephalopathy (ESB),
resulting in a mistrust of the consumer for the Belgian meat
and a lost of export dealings [1]. In order to restore the brand
image of Belgian meat products, it is important to assure a
traceability along the meat transformation chain. Traceability has been defined in ISO8042 as the ability to trace the
history, application or location of an entity by means of
recorded identification. In the context of food safety, trace* Corresponding author. Tel.: +32 4 366 39 73;
fax: +32 4 366 40 44.
E-mail address: bchina@ulg.ac.be (B. China).

ability is the ability to trace and follow a food, feed, foodproducing animal or substance intended to be, or expected to
be incorporate into a food or feed, through all stages of
production, processing and distribution (regulation 178/
2002/EC). In Belgium, this regulation was implemented
via several administrative traceability systems. The principal
is the SANITEL system including an automatic treatment of
data related to animal identification and registration. Moreover, the SANITEL system includes an individual identification of the animal by a number present on a earring [1].
The main disadvantage of this system is that, in the porcine
channel, the traceability stopped at the slaughterhouse. It is
therefore almost impossible to link a piece of meat with an
animal. Moreover, the administrative traceability is not
unfailing, the lost of documents and the risk of cheating
are real. For example, in case a sanitary embargo, to
guarantee the origin of the pork meat is essential. So, the

0379-0738/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2005.02.013

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F. Goffaux et al. / Forensic Science International 151 (2005) 239247

administrative traceability presents limits that the use of

genetic markers could overcome.
Indeed, the genome of individual animals differs from
each other (with the exception of monozygotic twins).
Moreover, each nucleated cell of an individual possesses
the same genome. Therefore, it is possible to distinguish all
individuals of the same species by analysing the genome of
any cell from any tissue since they possess differences in
their DNA sequence. Today, the genetic markers used for the
individual identification and parentage control are almost
exclusively microsatellite markers present randomly every
3046 kb in pig genome [2]. Single nucleotide polymorphism (SNP) markers are more abundant with an occurrence of
about one SNP per kb in human [3] and about one SNP per
500 base pair (bp) in mice [4] and cattle [5]. SNPs present the
following advantages over microsatellite markers: (1) relatively stable from generation to generation [6], (2) more
easily in laboratory handling and interpretation [6], (3)
usable for standardised representation of genotyping results
as a digital DNA signatures [7], and (4) compatible with
automation [8]. On the other hand, the most disadvantage is
that SNP are generally biallelic. Therefore, the information
content of SNPs is lower than the information content of
multiallelic (up to 10 alleles) microsatellites. But, this
disadvantage can be compensated by testing a higher number (at least 20) of markers [9].
The first step of this study was to find new SNPs present
in the 50 and 30 untranslated region (UTR) of pig genes. Next,
the more informative SNPs were selected to integrate a set of
SNP markers. After all, the potential utility of these markers
in animal identification was estimated. The final aim of this

work was to develop a powerful genetic traceability test

applicable to the major Belgian pig crossbred in order to
improve and to complement the used administrative system.

2. Material and methods

2.1. Animals and DNA samples
The panel of pigs consisted of 96 crossbred (Large
White  Pie train  Landrace) originated from five different
channels of pig production in Belgium and 32 purebred
(Belgian Pie train, Rehal Pie train, German Pie train, French
Landrace and BN K + Landrace) originated from the
C.I.A.P. (Interprofessional Center for Animals Promotion
and Improvement, Argenteau, Belgium). DNA was extracted
from pieces of cheek collected at the slaughterhouse and
from semen collected at the C.I.A.P., using the Wizard SV
Genomic DNA Purification System (Promega) following the
recommendation of the manufacturer.
2.2. PCR primer design
One hundred and forty primer pairs were designed for
PCR amplification and single strand conformation polymorphism (SSCP) analysis (not shown). Primers used for
the study of polymorphism rate and for the genotyping test
were listed on Table 1. All primers were designed with Oligo
6.6 software (Molecular Biology Insight). For SSCP analysis, PCR products length was comprised between 100 and
300 bp.

Table 1
PCR primers used in this study
GenBank accession no.

Primer

Sequence

Fragment
size (bp)

Annealing
temperature

AF329087

F5B
F6B

50 TCAGTACCCAGCCAGTT 30
50 GGGTTTTGAGTCTTACACTTG 30

365

58

AF342812

F17
F20

50 GCGCAGGGATGGTTTCAC 30
50 GGTGGCATCATGAGAACCTGA 30

422

61

AF451836

F47B
F50B

50 TGGCGTGTCTGAGTGCATCA
50 CCCTCTAGCCCTATCAAGTTCAA 30

407

58

AF139178

F53B
F54B

50 CAGCGAACCTCCACAGCATCT 30
50 CGGGATCCCAGACGAGA 30

401

63

AF038553

STAR1
STAR2

50 CCCAACCCACTGAGAGTGAAG 30
50 GGTCAAATCTGAGCCAAATCT 30

509

63

U12627

F89
F90

50 TGCCGAGTGACACAGATTTGT 30
50 AGTCACCAGTCAGGGCAATG 30

273

63

E01557

F103B
F104B

50 TAGGGCTAAAATGAATGT 30
50 TAACCCAAGTTTTGTGCTT 30

340

50

E08096

F317
F318

50 CCCAACCAGCAATATCATCAT 30
50 TTTCTTGGCTGTGCGACCTTA 30

314

58

F. Goffaux et al. / Forensic Science International 151 (2005) 239247

241

Table 1 (Continued )
GenBank accession no.

Primer

Sequence

Fragment
size (bp)

Annealing
temperature

AF034974

F119B
F120B

50 CCCACCTCGCATCACTCTAGCTG 30
50 GCGTCACAATTAAAGGAGCC 30

391

63

AF034974

F147
F148

50 GGCGTAGGAGTCCTCAG 30
50 CCTGCGAAATCGGAAAT 30

277

53

Y15710

F151B
F152B

50 AACACACAGTACACACAGCACGTAT 30
50 GCCAATTTAAAAGTCCCTAAG 30

380

55

AF426435

F165B
F166B

50 GCCGTGCTCCTCTGTAATTTG 30
50 GATCTTTGCCTCAGCCTGGAT 30

386

58

X98558

F243
F246

50 CTCTCCTTAATCCTGCGACCT 30
50 GCTTTAAATGGCCCTAGCGTC 30

493

64

AF020322

F249B
F250B

50 CCCTTTGGACAGACGCTTGAA 30
50 TTCTGGGTAGAGGGAAACCTG 30

497

58

AF327369

F277
F278

50 AGACTCCAGCCCAAACTG 30
50 CGGGTTGGAATGAATTTCT 30

214

58

AJ000928

CMYCF
CMYCR

50 ATCACTACTGCGGTACAACAG 30
50 GGATTTGGTGGTGGCAAGC 30

195

63

AJ493461

MYH4F
MYH4R

50 AGTGAAGAGTAATTCATCTAAA 30
50 GATTGCAAAATTCTCTGTAGA 30

124

55

AF473820

PKCTF
PKCTR

50 GATATCCTGAGACTTGCCTCT 30
50 GCGTCCACACATATTTCATT 30

364

61

Y16181

HFABPF
HFABPR

50 CTGCCCTAATCTGACCCTC 30
50 CAACAAGAACCGGAACTGAAC 30

241

58

AF227686

GNRHRF/2
GNRHRR/2

50 CCGAAATGGTAAACAGGGTGT 30
50 TCATATGGGCAGGGAGA 30

599

55

U70883

FUT1F
FUT1R

50 CATTGCCCACCTGTTCTTC 30
50 CTACCTACCGTTGGCAGTTTG 30

595

61

M29939

SLADQA152
SLADQA377

50 CGACCATGTTGCCTCCTA 30
50 CGCGGTGTTGTTGGAAC 30

238

55

X68247

RYR1336
RYR1810

50 TCTTGCCTCCGACTTCTCA 30
50 ACCGGAGTGGAGTCTCTGA 30

493

58

Y16180

F311
F312

50 TTTCACCAGACCCGATCATTC 30
50 GGACTCTGCCCTGTATTCCTA 30

592

63

AJ251197

F313
F314

50 GGGCAAGAGGAGAGGTAGCTC 30
50 GAGAGAAGGTCAGCCCAGAAT 30

548

58

2.3. PCR amplification

2.4. Single strand conformation polymorphism analysis

The PCR reactions contained 5 ml of purified DNA, 1 U of

Taq DNA polymerase (Amersham Biosciences), 5 ml of 2 mM
dNTP mix (Eurogentec), 5 ml of 10 PCR buffer and 0.5 ml of
each primer (40 mM) in a total volume of 50 ml. Reactions
were performed in a Mastercycler gradient (Eppendorf) with
the following conditions: initial denaturation at 94 8C for
5 min, followed by 35 cycles at 94 8C for 30 s, annealing
temperature (see Table 1) for 30 s and 72 8C for 1 min, and a
final extension at 72 8C for 5 min. PCR products were analysed
by 2% agarose gel electrophoresis in 1 TAE buffer.

For SSCP, 5 ml of the PCR product was mixed 1:1 with

denaturing buffer (95% formamide, 0.025% xylene cyanol
and 0.025% bromophenol blue), heat-denaturated at 95 8C
for 5 min, and then chilled on ice. Electrophoresis was
carried out at 12 8C constant temperature on a GeneGel
SSCP gel (Amersham Biosciences) in a GenePhor Electrophoresis Unit (Amersham Biosciences). The following conditions were used: 6 mA 90 V for 25 min and 14 mA 500 V
for 50 min. Bands were visualised with the DNA Silver
Staining Kit (Amersham Biosciences).

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F. Goffaux et al. / Forensic Science International 151 (2005) 239247

2.5. DNA sequencing

Amplification products were purified on a KingFisher
Magnetic Particle Processor (Thermo LifeSciences) using
the Wizard MagneSil PCR Clean-Up System (Promega).
Sequencing reactions were performed in 20 ml containing
5 ml of purified PCR product, 8 ml of DYEnamic ET Terminator sequencing premix (Amersham Biosciences) and
1 ml of primer (4 mM). Upper and/or lower PCR primers
were used as sequencing primer. The cycling conditions
were: 25 cycles at 94 8C for 20 s, 50 8C for 15 s, and 60 8C
for 1 min. Sequencing reactions were purified with AutoSeq96 (Amersham Biosciences) and automatic reading was
performed using a MegaBACE 500 DNA Analysis System
(Amersham Biosciences). The sequence data were analysed
with the Sequence Analyzer 3.0 software (Amersham Biosciences) and the sequence comparisons were performed with
ClustalW (http://www.ebi.ac.uk/clustalw/).
2.6. Single nucleotide primer extension (SNuPe)
The extension primers (Table 2) were selected according
to the guidelines recommended by Amersham Biosciences.
It included the absence of a G base in 30 .
Amplification products were purified as described in
DNA sequencing. For the primer extension reaction the
following mix was used: 5 ml of purified PCR product,

1 ml of extension primer (2 pmol) and 4 ml of SNuPe premix

(Amersham Biosciences) in a total volume of 10 ml. Following conditions were applied: 25 cycles at 96 8C for 10 s,
50 8C for 5 s and 60 8C for 10 s. Reaction products were
purified as described in DNA sequencing. Finally, 5 ml of the
reaction products were combined with 5 ml the multi-injection marker for separation and detection on the MegaBACE
500 capillary electrophoresis system. Data were analysed
with SNP Profiler 1.0 software (Amersham Biosciences).
Extension primers were presented on Table 2.

3. Results
3.1. PCR amplification and SSCP analysis
In order to find new SNPs, a first screening using the
SSCP method was performed on 20 50 UTR and 42 30 UTR
from GenBank. One hundred and forty primer pairs were
used to amplify 20 different DNA samples. The resulting
amplicons were analysed by SSCP. Thirty-nine primer pairs
of 140 did not give the expected result by PCR (no amplification or non-specific amplifications). A polymorphism in
the SSCP migration profile was observed for 38 out of 101
different analysed amplicons. Presence of SNP(s) was confirmed or invalidated in these PCR products by DNA
sequencing.

Table 2
Extension primers used for the SNuPe reaction
GenBank accession no.

SNP
Type

Primer

Sequence

Sense

Position

AF329087

Y
S

618
642

619L21
621U21

50 TTGAGTTAGGACCACGAT 30
50 CGTGGTCCTAACTCAATTGGA 30

Reverse
Direct

AF451836

Y
H
K

139
159
317

118U21
160L21
318L21

50 CCCTTTAGGTCTCAATTTCCT 30
50 GTCAGGTCATCCGCAATCCTC 30
50 GTTTTCCCAAGGCCACACAGA 30

Direct
Reverse
Reverse

AF038553

308

287U21

50 TAGAAAGAAAAGCAGAAAATC 30

Direct

U12627

K
R
V

88
208
251

89L18
209L19
233U18

5 GCTCATAGGAACACAGAC 3
50 CCGCCTGCCTCCCCCCAAC 30
50 GACCATCTCCATCCTTAT 30

E08096

1350

1351L18

50 AACACAAGGATCTGGATA 30
0

Reverse
Reverse
Direct
Reverse
0

AF034974

R
Y
R

848
864
994

232U21
270L21
400L21

5 CCCTTCGCGGCGTTACTCAGC 3
50 GACCAAGCCCAGTCATGAAAC 30
50 CCAATTCTTTCCTGTGGCGTA 30

Direct
Reverse
Reverse

AF426435
AF020322
AF473820
U70883
M29939
X68247
Y16180
AJ251197

Y
Y
R
Y
Y
Y
R
Y

501
615
224
2148
245
1666
1929
384

502L21
284U21
225L22
2129U19
224U21
COP5
1930L21
363U21

50
50
50
50
50
50
50
50

Reverse
Direct
Reverse
Direct
Direct
Reverse
Reverse
Direct

CCTACCCATCAAGCCAGTGGA 30
ATTATTTTCAGAGGAAAAAGT 30
TCTTTTCCAGGTTGAAAAGGAA 30
GCAAGCACTCACCAACCCC 30
TGATGGCGACGAGGAATTCTA 30
ATGAGATCTTGGTTGGAGC 30
ACCGTGACTGAGCAGGCTTAA 30
GTAACACCTTGGGCAAGTCAC 30

F. Goffaux et al. / Forensic Science International 151 (2005) 239247

3.2. Polymorphism identification

In order to determine the type and the position of the
SNP(s), PCR products presenting a SSCP polymorphism

243

were submitted to sequencing on both strands. Presence of

SNP was confirmed for only 18 PCR products out of 38
different polymorphic amplicons. In total, 39 new SNPs
were discovered (Table 3), and 1 SNP identified in the

Table 3
Type and position of SNPs
GenBank accession no.

Chr.

SNP
Position

Allele frequency

Variation

Allele 1

Allele 2

Allele 3

AF329087 (50 UTR)

618
642

Y
S

0.67
0.4

0.33
0.6

0.44a
0.48a

AF342812 (30 UTR)

116
375

M
Y

0.1
0.15

0.9
0.85

0.17
0.26

AF451836 (50 UTR)

103
139
159
317

R
Y
H
K

0.84
0.8
0.19
0.25

0.16
0.62
0.33
0.75

0.27
0.47a
0.63a
0.38a

0.48

AF139178 (50 UTR)

393

0.95

0.05

0.10

AF038553 (50 UTR)

15

308
338
388
397
431
495
553
579
614
615
621

R
R
W
Y
Y
Y
Y
S
Y
R
Y

0.73
0.73
0.85
0.73
0.875
0.85
0.875
0.85
0.85
0.87
0.88

0.27
0.27
0.15
0.27
0.125
0.15
0.125
0.15
0.15
0.13
0.12

0.39a
0.39
0.25
0.39
0.22
0.25
0.22
0.25
0.26
0.23
0.21

88
199
208
251

K
S
R
V

0.62
0.5
0.26
0.57

0.38
0.5
0.74
0.29

0.47a
0.5
0.39a
0.58a

E01557 (30 UTR)

2989

0.98

0.02

0.04

E08096 (30 UTR)

1350

0.73

0.27

0.39a

U12627 (30 UTR)

0.14

AF034974 (30 UTR)

848
864
994
2284

R
Y
R
R

0.62
0.79
0.74
0.27

0.38
0.21
0.26
0.73

0.47a
0.33a
0.39a
0.4

Y15710 (30 UTR)

1420
1486

Y
R

0.85
0.86

0.15
0.14

0.25
0.23

359
390
501

S
Y
Y

0.17
0.16
0.18

0.83
0.84
0.82

0.28
0.27
0.30a

1306
1324
1524

R
Y
Y

0.66
0
0.65

0.34
0
0.35

0.45b
0b
0.45

615
739

Y
Y

0.67
0.84

0.33
0.16

0.44a
0.26

1947

0.98

0.02

0.04

AF426435 (50 UTR)

X98558 (50 UTR)

AF020322 (30 UTR)

AF327369 (30 UTR)

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F. Goffaux et al. / Forensic Science International 151 (2005) 239247

Table 3 (Continued )
GenBank accession no.

Chr.

AJ000928 (50 UTR)

SNP

Allele frequency

Position

Variation

Allele 1

Allele 2

Allele 3

1189

0.97

0.03

0.06b

AJ493461 (30 UTR)

12

26

0b

AF473820 (30 UTR)

10

171
222
224
339

Y
K
R
Y

0
0.67
0.67
0.04

1
0.33
0.33
0.96

0b
0.44b
0.44a,b
0.08b

Y16181 (30 UTR)

310

0b

AF227686 (30 UTR)

661
755
1632
1721

Y
K
W
S

ND
ND
1
0.07

ND
ND
0
0.93

NDb
NDb
0b
0.13b

U70883 (30 UTR)

915
1465
2148

R
R
Y

ND
ND
0.64

ND
ND
0.36

NDb
NDb
0.46a,b

245
254
278
279
293
294
296
301
308
314
332
339
342
352
353
363
364
368

Y
R
M
R
Y
R
W
S
W
R
Y
R
K
M
Y
Y
S
Y

0.75
0.94
0.93
0.5
0.64
0.38
0.98
0.98
0.96
0.96
0.62
0.9
0.78
0.89
0.92
0.88
0.81
0.87

0.25
0.06
0.07
0.5
0.36
0.62
0.02
0.02
0.04
0.04
0.38
0.1
0.22
0.11
0.08
0.12
0.19
0.13

0.38a,b
0.11b
0.13b
0.5b
0.46b
0.47b
0.03b
0.03b
0.07b
0.07b
0.48b
0.1b
0.34b
0.19b
0.15b
0.22b
0.32b
0.22b

M29939

X68247

1666

0.45

0.55

0.44a,b

Y16180

647
737
861
1489
1776
1811
1929
1970
2767

Y
Y
M
Y
K
S
R
Y
Y

ND
ND
ND
0.07
0.6
0.58
0.61
0.61
ND

ND
ND
ND
0.93
0.4
0.42
0.39
0.39
ND

NDb
NDb
NDb
0.12b
0.48b
0.49b
0.47a,b
0.47b
NDb

AJ251197

202
254
384
438

R
R
Y
R

0.47
0.44
0.48
0.47

0.53
0.56
0.52
0.53

0.5b
0.49b
0.5a,b
0.5b

M = A or C, R = A or G, W = A or T, S = C or G, Y = C or T, K = G or T, H = A or C or T, V = A or C or G; ND: not determined.

a
SNPs selected.
b
SNP present in GenBank.

F. Goffaux et al. / Forensic Science International 151 (2005) 239247

50 UTR of the heart fatty acid-binding protein (H-FABP)

gene was already described (GenBank X98558). So, over
23,382 bp amplified and sequenced, 40 SNPs were identified, representing an average of 1 SNP per 585 bp. A 509 bp
fragment of the 50 UTR of the steroidogenic acute regulatory
(StAR) protein gene (GenBank AF038553) contained 11
SNPs, and a new SNP (at position 1524) was identified in the
50 UTR of the H-FABP gene. Thirty SNPs of 40 were
transitions and 8 were transversions, and 2 polymorphisms
showed more than two alleles.
3.3. Determination of the polymorphism rate
In order to determine the polymorphism rate of our 40
SNPs, the DNA regions including SNPs were amplified by
PCR and sequenced. Forty-eight different pig DNA samples were used. For each SNP, allelic frequencies were
estimated and the heterozygosity (H) was calculated as
follow:
n
X
H
i p2i :
i1

Results are presented on Table 3. A SNP was considered

as highly informative when the H value was higher than 0.3.
Only 21 SNPs out of 40 presented this criterion. Nevertheless, SNPs in the same sequence were sometimes highly
associated, and in this case only one of them was selected.
Therefore, to obtain a minimum of 20 highly informative
SNPs usable in a genotyping test, our SNPs panel was
completed with SNPs previously identified SNPs present
in GenBank. Polymorphism rate for these SNPs was also
determined (Table 3). For four SNPs, no polymorphism was
observed in the DNA sequence of the tested samples. After
this first discrimination based on the H value, 22 SNPs
remained available (16 newly described and 6 previously
described).

245

3.4. Probability of identity in crossbred pig population

and genotyping assay
The SNPs genotyping method chosen was primer extension. Due to constraints in the extension primer design, for
some SNPs no extension primer could be selected (i.e. in
X98558 sequence). Finally, 21 SNPs remained usable and
were included in the genotyping test (Table 3). The capacity
of the 21 SNPs panel to identify all animals in a population
was evaluated by primer extension 96 individuals representing the major crossbred pig population in Belgium and 32
purebred individuals. The average for the H value for these
21 SNPs was 0.44. The probability that an individual A
selected randomly in a population is identical to an individual B selected randomly in the same population was
calculated as follow [10]:
!
ni
nX
ni
r
i 1 X
Y
X
4
2 2
PA B
qi j 4
qi j qik
i1

i1

j1 k j1

This probability is approximately 7  109 for the 21 SNPs

selected.
Results showed that all tested pigs possessed a different
genotype (data not shown) indicating that our test was
discriminant.
3.5. Application in food traceability
In the administrative traceability system, each individual
harboured a earring with a number allowing to identify the
animal and the farm of origin. Therefore, an ear sample and a
meat samples were sampled on two carcasses. Moreover, in
order to introduce controls, five different ear samples and
five different meat samples were also sampled at the
same moment in the same slaughterhouse. The DNA of
the 14 samples was extracted and the genotyping procedure
was applied. The results were summarised on Table 4.

Table 4
Results of the genotyping procedure
Sample number

Animal number

Tissue

Genotype codea

Result

1
2
3
4
5
6
7
8
9
10
11
12
13
14

1
1
2
2
3
4
5
6
7
8
9
10
11
12

Ear
Meat
Ear
Meat
Ear
Ear
Ear
Ear
Ear
Meat
Meat
Meat
Meat
Meat

133432121131132220303
133432121131132220303
213431222121132212323
213431222121132212323
100402001210131220013
103332222131232213212
132431222131132112223
133333222101232123212
133432121113113220303
133433222231133212322
210402002101133112331
213431222231133222323
212522323121133122321
113432222121212122212

Identical to sample 2
Identical to sample 1
Identical to sample 4
Identical to sample 3
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample
No identical sample

a
For biallelic marker: 0 = no result, 1 = homozygote 1; 2 = homozygote 2, 3 = heterozygote; for triallelic marker: 0 = no result, 1 = homozygote 1, 2 = homozygote 2, 3 = homozygote 3; 4 = heterozygote 1, 5 = heterozygote 2; 6 = heterozygote 3.

246

F. Goffaux et al. / Forensic Science International 151 (2005) 239247

The genotyping procedure allowed to reconstitute the two

right ear-meat pairs. Therefore, it was possible to establish
an objective link between an individual piece of meat, the
animal and the farm.

4. Discussion
The recent crisis in the animal production area emphasised the need of an improved animals and animal products
identification system that can guarantee the traceability from
the producer to the consumer. Moreover, in case of transmissible diseases that are of major importance in the international trade of animals and animal products, safety devices
are taken, as a consequence a considerable financial lost for
producers. Therefore, cheaters could attempt to falsify the
meat origin. In order to avoid these problems, a powerful
traceability system is required.
A pig carcass has a unique identity, linked to the identity
of the live animal. However, after processing from the
slaughterhouse to the retail point, the carcass may be disassembled into a lot of separate pieces. To maintain identity
through this processing and distribution chain using conventional labelling system is difficult [11]. Only DNA could
help the administrative traceability as DNA sequence in
each nucleic cell of an individual is identical and this
sequence is specific to the individual (excepted for monozygotic twins).
To develop a traceability test in pig based on single
nucleotide polymorphisms, two choices presented to us: (i)
working with SNPs already described in GenBank or (ii)
searching for new SNPs in the major crossbred pig population
in Belgium. The second solution was first investigated. We
studied 50 and 30 UTR of pig genes by PCR amplification and
SSCP analysis. Seventy-two percent of the primer pairs
designed gave the expected PCR product. For the others,
either PCR conditions used were not optimal, or primers
annealed in regions where DNA forms secondary structures,
or primers were not specific for the region to amplify. Migration polymorphisms were observed for only 38% of the primer
pairs tested. Presence of SNPs was confirmed by sequencing
in only 18% of the tested amplicons.
In total, we identified 40 SNPs, representing an average
of 1 SNP per 585 bp. This value is low compared with a
similar study [12]. By directly sequencing PCR amplification products from genes on the porcine chromosome 2,
these authors found 1 SNP per 108 bp. This strong difference
could be explained by the fact that we probably missed SNPs
when performing a first screening by SSCP analysis. Interestingly, the first third of the 50 UTR of the StAR protein gene
contained 11 SNPs representing an average of 1 SNP per
46 bp. Such regions with a high concentration of SNPs were
already described in others porcine genes [13,14]. Composition of the 39 new SNPs identified in this study was 74% C/T
or A/G, 21% A/T or G/C or A/C or G/T, and 5% others. This
observation is comparable with previous studies [15,12].

The heterozygosity values for each new SNP and for

SNPs previously described was calculated. The H value
varied from 0 to 0.63. The H values >0.5 were explained
by the fact that two SNPs showed more than two alleles.
Furthermore, some SNPs previously described were not
observed in the pig population used in this study. This
observation underlined the fact that SNP could be population
specific. Twenty-one informative SNP markers (H > 0.3)
were selected for the genotyping test.
The probability that two individuals selected in the same
population are identical was approximately 7  109. Since
the Belgian pig population is 7  106, the test was considered as sufficiently discriminant. The genotype of 96 individuals originated from five different pig production
channels in Belgium and of 32 purebred individuals kept
for breeding was performed by primer extension. All individuals possessed a unique DNA fingerprinting.
This genotyping procedure was applied to demonstrate
the ability to find back the link between a piece of meat and
the animal of origin. Among, 14 tissue samples, two pairs
were present. Using our genotyping procedure, it was possible to reconstitute the two pairs.
Nevertheless, this procedure is only applicable to pure
tissue. In meat mixtures such as minced meat, the presence
of several individuals will give results very difficult to
analyse due to the stacking of the signals.
Therefore, it seems that our genotyping test might be
usable (to a certain extent) for a genetic traceability system.
Such a system could be used, together with an administrative
one, to avoid frauds during a crisis and to improve traceability of pigs. In this context, the constitution of a databank
including the genotype of each individual could be useful.

Acknowledgements
This work was financed by the Direction Ge ne rale des
Technologies, de la Recherche et de lEnergie (DGTRE) du
Ministe`re de la Re gion Wallonne (convention no. 114880).

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