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Meat Science 76 (2007) 666674
www.elsevier.com/locate/meatsci
Abstract
We evaluated and compared the utility of mitochondrial markers viz. 16S rDNA and NADH dehydrogenase subunit 4 (ND4) and a
nuclear marker viz. the actin gene to identify the specimens of animal origin for forensic identication, food regulatory control and to
prevent illegal trading, poaching and conservation of endangered species. We also tested PCR ngerprinting methods like RAPD and
actin barcoding to generate species-specic ngerprints. Our results suggested that mitochondrial markers are more ecient than
nuclear markers for the purpose of species identication and authentication. Among PCR ngerprinting approaches, RAPD was proved
to be more discriminatory, accurate and ecient than actin ngerprinting. Considering the present scenario in trading of vertebrate animal tissues like bualo, cow, pig, goat, chicken, frogs, shes and snakes etc., mitogenomics based technology proved to be ecient and
reliable in resolving problems like meat adulteration and smuggling across countries.
2007 Elsevier Ltd. All rights reserved.
Keywords: Mitochondrial markers; Nuclear marker; 16S rDNA; ND4; RAPD; Meat
1. Introduction
Reliable labeling of meat products oered for sale is
important in order to assure the consumers about the identity and quality of the food products they consume for a
variety of reasons including adulteration and socio-religious
factors. Due to an increase in the consumption demand and
high cost, other cheaper animals are being used as fraudulent substitutes or mislabeled. When an endangered animal
has been killed for food or sport, phenotypic markers are
often destroyed or intentionally removed to create problems
for personnel who are involved in determining the species
origin of an animal or products derived from it in order to
enforce conservation and or health-related regulations
(Palumbi & Cipriano, 1998). Curbing the illegal trading of
ecologically and economically important animals is neces*
0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2007.02.006
products. One of the stages of dealing with biological materials submitted to forensic laboratories is species identication of the biological evidence e.g. blood strains on a cloth
collected from a fatal road accident and insurance claims
that involve road accidents with animals require correct
species identication. Mitochondrial DNA (mt-DNA)
forensic analysis can be used in such disputes even without
the reference sample. The characteristic high copy number,
maternal inheritance and high degree of sequence variability make mt-DNA a powerful tool for forensic identication. Most of these methods make use of PCR
amplication and direct sequencing of a conserved gene
from the sample e.g. mt-12S rDNA (Rastogi et al., 2004),
mt-16S rDNA (Borgo, Souty Grosset, Bouchon, & Gomot,
1996) cytochrome b gene (cytb) (Wong et al., 2004), use of
oligonucleotide probes (Hunt, Parkes, & Lumley, 1997),
amplication of species-specic DNA sequences (Arevalo,
Davis, & Sites, 1994; Meyer, Hofelein, Luthy, & Candrian,
1995), microsatellites analysis (Lorenzini, 2005), RFLP
analysis (Meyer et al., 1995), SSCP of PCR amplied fragments (Cespedes et al., 1999) and RAPD (Calvo, Zaragoza,
& Osta, 2001; Franklin, Taylor, & Mills, 1999). The denition of mono-locus-specic primers is quite reliable but the
technique is conditioned by a prior knowledge of nucleotide sequences anking the loci and richness of gene databases. The advantage of RAPD ngerprinting lies in the
fact that it is a sequence independent approach and each
primer DNA annealing will produce a dierent spectrum
of fragments from the PCR generating a species-specic
ngerprint. RAPD is faster and cheaper than the other
DNA based methods, but major the drawback is the poor
reproducibility of the results obtained (Louie et al., 1996;
Welsh & McClelland, 1990). The 16S rDNA and ND4
genes have been very well studied among dierent vertebrate groups at the population level (Goebel, Donnelly,
& Atz, 1999; Hillis, Mable, & Moritz, 1996; Simon et al.,
1994). These studies have indicated that the level of 16S
rDNA and ND4 gene sequence variation is suitable for
addressing general questions on interspecic diversity. To
date, no studies have been reported using the ND4 gene
sequences to identify the species origin of animal tissues.
To the best of our knowledge, this is the rst report where
ND4 gene sequences were used for the purpose of species
identication of animal tissues.
Reliance on a single, maternally inherited character (mtDNA) can provide misleading results in situations where
there is a possibility of hybridization or introgression,
and analyses of both nuclear and mitochondrial markers
are therefore desirable (Palumbi & Cipriano, 1998). Several
nuclear genes, which are conserved throughout the vertebrates and invertebrates like actin and histones, have been
reported in investigations involving adulteration and conservation issues (Fairbrother, Hopwood, Lockley, &
Bardsley, 1998; Svenson & Whiting, 2004). Genome
sequencing projects on many vertebrates revealed high
sequence homology in protein coding regions of actin genes
but considerable variability in intron number and sizes.
667
668
to check the consistency of results. All PCRs were performed at least on two dierent occasions in order to check
the reproducibility of the experimental procedure and the
ngerprint patterns. A negative control was also included
in all PCRs. PCR products were then puried for sequencing by the polyethylene glycolNaCl precipitation method
as described earlier (Rastogi et al., 2004). Puried PCR
products (100 ng) were directly sequenced using a 3730
DNA Analyzer (Applied Biosystems, Foster City, CA) as
per the manufacturers protocol. For RAPD, only single
primer was added in the PCR reaction. We have tested ve
dierent RAPD primers (Operon Technologies) for their
ability to provide readily interpretable and reproducible
RAPD proles. Only one primer was found to give a clear
and distinct RAPD ngerprint, and was used in further
investigations. In order to generate species-specic actin ngerprints, we amplied introns of the actin gene, which separate exon 6 and 7. Simultaneously, we made slight
modications to the PCR conditions (Table 1) in order to
get amplication of a single intron generating a single band
of 400 bp, which can be sequenced directly.
2.3. Dendrogram construction
PCR ngerprints were interpreted after normalization
and dendrograms were constructed for each ngerprinting
method using the UPGMA algorithm for cluster analysis
by Quantity One-4.4.0 gel documenting software (Bio-
Table 1
PCR primers and amplication conditions
Gene targeted
Primer sequence (5 0 3 0 )
mt-16S rDNA
16SAT
CGCCTGTTTATCAAAAACA
16SBT
CCGGTCTGAACTCAGATCACGT
mt-ND4
ND4
CATTACTTTTACTTGGATTTGCACCA
LEU
CACCTATGACTACCAAAA
GCTCATGTAGAAGC
ActinF
CCTACAACAGCATCATGAAGTG
ActinR
GCTGATCCACATCTGCTGGAAG
Actin (for barcoding)
ActinF
CCTACAACAGCATCATGAAGTG
PCR conditions
9
94 C; 1 min =
55 C; 1 min 35X
;
72 C; 1 min
9
94 C; 1 min =
55 C; 1 min 35X
;
72 C; 1 min
Reference
535
700
400
9
95 C; 2 min =
45 C; 2 min
25X
;
68 C; 10 min
95705
9
95 C; 1 min =
36 C; 1 min 40X
;
72 C; 2 min
1761250
9
95 C; 0:30 min =
50 C; 0:45 min 25X
;
72 C; 0:30 min
ActinR
GCTGATCCACATCTGCTGGAAG
RAPD
F4
GGTGATCAGG
X indicates number of cycles in PCR. All PCRs included an initial denaturation at 94 C for 3 min and nal extension at 72 C for 10 min
Rad, Hercules, CA). Computed similarities among ngerprints were estimated by means of the dice similarity coefcient. To survey the phylogenetic utilities of the dierent
markers, we used nucleotide sequence data for further phylogenetic analyses. The electropherograms obtained for
both the strands were checked by BioEdit (Brown, 1999)
and also by manual proof reading for any ambiguity or
inframe errors. Sequences were aligned using the Clustal
W program (Thompson, Higgins, & Gibson, 1994). Similarity searches for obtained partial sequences were carried
out using BLAST at NCBI (Altschul, Gish, Miller, Myers,
& Lipman, 1990). Ambiguously aligned portions were
deleted from the beginning and end using DAMBE (Xia,
2000). Phylogenetic trees were constructed by UPGMA
method using Molecular evolutionary genetic analysis
(MEGA) v3.1 (Kumar, Tamura, & Nei, 2004). Bootstrap
re-sampling analysis for 1000 replicates was performed to
estimate the condence of tree topologies. Gaps/missing
nucleotide positions in alignment were completely deleted.
Similarity tables showing percentage relatedness among
gene sequences were drawn for each marker using the
DNADIST program of PHYLIP (Felsenstein, 1989).
3. Results and discussion
Surveillance to detect adventitious or fraudulent contamination in meat products serves to reassure all parties
within the food supply chain. This is particularly true of
ethnic groups whose religious beliefs forbid the consumption of particular species and who require assurance that
they are purchasing food that conforms to their beliefs.
The ability to intercept adulterated meat before it enters
domestic commerce is a high priority issue. Short DNA
sequences from a dened stretch of the genome provide a
DNA barcode for identifying species in question. To use
this approach on a large scale, construction of DNA barcodes for each species will provide a key for identifying
unknown species. The eciency of such a DNA based
identication system is promising with increasing taxon
coverage of gene databases and availability of faster and
cheaper sequencing techniques. To be eective, a diagnostic
barcode must demonstrate consistent dierences among
closely related species and exhibit very limited intraspecic
variation. The objective of the present study was to develop
a key for the identication of tissue samples based on independent mitochondrial and nuclear markers.
3.1. Species identication by PCR sequencing approaches
3.1.1. Mitochondrial markers (16S rRNA and ND4 gene)
On the basis of partial 16S rDNA sequence data, all tissue samples (bualo, cow, chicken, pig, goat, frog, sh and
snake) showed a high degree of similarities to corresponding sequences in GenBank database and were perfectly
identied up to species level. Thus, the partial 16S rDNA
sequences can be used as a suitable DNA barcode for species identication and authentication of animal tissues.
669
Buffalo
89
Cow
91
100
Pig
Goat
79
Fish
83
Frog
Snake
Chicken
50
40
30
20
10
670
Table 2
Similarities for the sequences of mt-16S rDNA, mt-ND4 and nu-actin gene
Specimen used for analysis
Gene amplied
Accession numbera
Accession number
Score (bit)
E-value
16S rRNA
ND4
Actin
DQ904379
DQ904397
DQ904371
AF547270
AY488491
U02285
1045
1620
440
0.0
0.0
3e120
16S rRNA
ND4
Actin
DQ904381
DQ904390
DQ904372
AF492350
AY126697
BC102992
932
700
1108
0.0
0.0
0.0
100
94
98
16S rRNA
ND4
Actin
DQ904380
DQ904391
DQ904373
DQ648776
X52392
AF012348
1110
724
458
0.0
0.0
1e125
100
97
94
16S rRNA
ND4
Actin
DQ904382
DQ904398
DQ904374
AF486874
DQ518915
U16368
896
1241
446
0.0
0.0
5e122
98
96
94
16S rRNA
ND4
Actin
DQ904383
DQ904395
DQ904375
AF533441
AF533441
U02285
331
1124
416
7e88
0.0
2e113
92
100
95
16S rRNA
ND4
Actin
DQ904384
DQ904392
DQ904376
AY458592
AY458592
AB092520
880
2516
618
0.0
0.0
3e174
97
99
98
16S rRNA
ND4
Actin
DQ904389
DQ904393
DQ904378
AY458877
AP006739
AF393832
952
2664
1982
0.0
0.0
0.0
97
99
97
16S rRNA
ND4
Actin
DQ904385
DQ904396
DQ904377
AY701032
AB179619
AY734452
797
365
1255
0.0
2e97
0.0
96
83
99
16S rRNA
ND4
DQ904386
DQ904394
DQ530062
U20753
733
2633
0.0
0.0
100
99
16S rRNA
ND4
DQ904386
DQ904394
Panthera pardus
Felis catus
DQ530062
U20753
839
2633
0.0
0.0
100
99
16S rRNA
ND4
DQ904386
DQ904394
Panthera pardus
Felis catus
DQ530062
U20753
906
2633
0.0
0.0
99
99
Percentage similarity
99
98
95
Blast results indicating the similarity of each species included in the study with closest known species in the database, with bit-scores and E-value for each mitochondrial and nuclear markers. Bracketed
denotion in front of species name indicates family name for the particular specimen tested.
a
Accession numbers generated in this study.
100
Cow
100
87
Pig
100
Chicken
Snake
Frog
Goat
94
Fish
140
120
100
80
60
40
20
100
671
product and making the technique simple for routine analysis. Species identication using intron sequences present
between exon 6 and 7 of actin gene was not found to be satisfactory because only chicken and pig samples were identied perfectly up to species level using this nuclear marker.
The poor identication based on this gene was again attributed to non-availability of the actinintron sequences of B.
bubalis, B. indicus, C. hircus, B. melanostictus, Mystus sp.,
and Uropeltis sp. in GenBank for similarity studies. Phylogenetic analysis based on intron sequences identied two
clusters A and B at 70% similarity (Fig. 4). Cluster A
was further subdivided into three subclusters I, II, III. Subcluster I contained three samples bualo, cow and pig that
were related to each other at similarity ranging from 48
98%. Subcluster II included only the chicken sample which
showed similarity of 3256% with other samples, while sub
cluster III contained only the goat sample with 3259%
similarity with other samples. Cluster B contained three
samples frog, sh and snake that were related to each other
with similarity ranging from 83% to 89%.
3.1.2.1. Nucleotide accessions numbers. The nucleotide gene
sequences generated under this study for 16S rDNA, ND4
and actinintron genes were deposited in GenBank under
accession numbers DQ904379DQ904389, DQ904390
DQ904398 and DQ904371DQ904378 respectively.
3.1.3. Species identication by PCR ngerprinting
Both RAPD and actin ngerprinting methods allowed
dierentiation among tissue samples. RAPD ngerprinting
resulted in relatively simple DNA ngerprints from which
the species origin could be visually inferred, making the
technique especially suitable for routine analysis. The
application of actin barcoding also allowed the discrimination among dierent tissue samples. However, it gave a
higher number of bands, including a signicant number
of faint bands, which made visual interpretation a little difcult. Although the unique properties of the actin multigene family make it attractive for further authentication
studies, the low template copy number per cell is disadvantageous in terms of sensitivity, compared to more abundant
mt-DNA templates (Fairbrother et al., 1998).
Cow
100
35
Pig
Chicken
Goat
Frog
100
Snake
87
Fish
30
20
10
The discriminating ability of RAPD-PCR assay is virtually unlimited as it is always possible to use other random
primers. In the present study, we tested ve primers
(Operon I. D. C4, D5, F4, F7 and F5) and, nally one of
them (F4) was selected for RAPD ngerprinting on the
basis of number, intensity and distribution of bands to
clearly discriminate various species. Our data clearly
showed that in each case primer F4 was able to generate
characteristic species-specic ngerprints. RAPD ngerprinting with primer F4 allowed the discrimination among
bualo, cow goat, pig and chicken tissue (Fig. 5). The analysis has been repeated twice on two dierent occasion and
672
Fig. 5. RAPD analysis using the primer F4. Fingerprints were visualized
on 2% agarose gel. Lane 1. bualo, Lane 2. chicken, Lane 3. cow, Lane 4.
goat, Lane 5. pig, Lane 6. 1Kb plus DNA ladder (Invitrogen).
Table 3
Similarity matrix based on RAPD ngerprinting data showing percentage
relatedness among tissue specimens
Specimen
Cow
Chicken
Bualo
Pig
Goat
Cow
Chicken
Bualo
Pig
Goat
100
17.6
18.1
15.9
17.3
100
2.9
2.9
7.1
100
28.1
15.7
100
24.3
100
673
Fig. 7. Actin ngerprinting using the primers targeting the intron region
present between exon 6 and 7 of multiple actin genes. The ngerprints
were visualized on 2% agarose gel. Lane 1. bualo, Lane 2. chicken, Lane
3. cow, Lane 4. goat, Lane 5. pig, Lane 6. 1Kb plus DNA ladder
(Invitrogen).
Table 4
Similarity matrix based on actin ngerprinting data showing percentage
relatedness among tissue specimens
Specimen
Bualo
Chicken
Bualo
Chicken
Cow
Goat
Pig
100
19.9
40
46
21.8
100
13.6
39.4
49.2
Cow
Goat
Pig
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38.4
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