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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
240
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
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
242
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
243
Table 3
Type and position of SNPs
GenBank accession no.
Chr.
SNP
Position
Allele frequency
Variation
Allele 1
Allele 2
Allele 3
618
642
Y
S
0.67
0.4
0.33
0.6
0.44a
0.48a
116
375
M
Y
0.1
0.15
0.9
0.85
0.17
0.26
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
393
0.95
0.05
0.10
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
2989
0.98
0.02
0.04
1350
0.73
0.27
0.39a
0.14
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
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
244
Table 3 (Continued )
GenBank accession no.
Chr.
SNP
Allele frequency
Position
Variation
Allele 1
Allele 2
Allele 3
1189
0.97
0.03
0.06b
12
26
0b
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
310
0b
661
755
1632
1721
Y
K
W
S
ND
ND
1
0.07
ND
ND
0
0.93
NDb
NDb
0b
0.13b
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
245
i1
j1 k j1
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
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].
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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