Genetic Testing for Porcine Stress Syndrome Using Mutagenically Separated-Polymerase Chain Reaction

Abstract

Porcine Stress Syndrome (PSS) is a defect in the Halothane (Hal) gene that produces pale, soft and exudative meat of inferior quality that results to significant losses in the meat industry. This study was conducted to detect PSS in pigs from seven farms in Luzon, Philippines which are used for breeding purposes. They were classified as normal (NN), stress carrier (Nn) and mutant (nn). This classification will help to form a new breeding system to be developed ensuring that all offspring are free of the stress gene. Characterization of the Hal gene was done by collecting blood samples subjected to DNA extraction and genotyping using mutagenically separated-polymerase chairn reaction (MS-PCR) which is an optimized one step process of PSS detection. Out of 427 samples, 22 were found to be mutant, 34 were carrier, and 371 were normal. Results for genotypic frequency showed that 87% pigs are normal (NN); 8% are heterozygotes (Nn) and only 5% are stress-positive (nn). Results were validated through DNA sequencing which showed the same results with MS-PCR. A genetic screening using this developed method for the Philippine setting is recommended to be able to minimize the effect of PSS.

Full text

Genetic Testing for Porcine Stress Syndrome Using Mutagenically
Separated-Polymerase Chain Reaction
Jessica G. Manalaysay1, BS, Claro N. Mingala DVM, MVSc, PhD1*,
Domina Flor L. Gamboa, BS2, Rubigilda Paraguison-Alili BS, MS, PhD1
and Jocelyn G. Rafanan BS, MS2
1
Philippine Carabao Center National Headquarters and Genepool, Munoz, Nueva Ecija,
Philippines;
2
University of the Philippines Baguio, Baguio City, Benguet, Philippines
Philipp. J. Vet. Med., 51(2): 125-130, 2014
Halothane gene has been identied
as the one responsible for porcine stress
syndrome (PSS) elicited when the animals are
experiencing stress or when they have been
exposed to halothane (an anesthetic gas), thus,
the name “halothane gene” (Rosenvold and
Andersen, 2003). It has two possible alleles,
one dominant (N) and one recessive (n) and
is located at a single locus only which occurs
in three possible genotypes: homozygous
dominant (normal), NN; heterozygous
dominant/carrier, Nn; and recessive/ positive,
nn. Pigs that inherit the recessive n allele will
be more susceptible to stress and will be more
likely to develop pale, soft and exudative (PSE)
pork postmortem (Du, 2004).
According to Stalder and Conatser
(1999), PSS is caused by a defect in the
ryanodine receptor 1 (RYR1) gene located at
the chromosome 6 of pigs (2n=38). The defect
is caused by Cytosine/Thymine mutation
at nucleotide 1843 in the RYR1 gene. The
structural change in calcium ion release
channel/RYR1 coded by the RYR1 loci allelic
genes is considered to be the reason for PSS
(Jovanovic et al., 2005). Pigs with PSS may
suffer the following symptoms: heat stress,
labored breathing, muscle rigidity and in
worst cases, death. Though PSS is thought to
be a characteristic of domesticated pigs, the
incidence of this condition may vary in between
breeds as well as in the country of origin (Basic
et al., 1997). Frequencies of the genotypes
may also differ from one breed to another as
described in the study of Bastos et al. (2000).
*FOR CORRESPONDENCE:
(email: cnmingala@hotmail.com)
RESEARCH NOTE
ABSTRACT
INTRODUCTION
Porcine Stress Syndrome (PSS) is a defect in the Halothane (Hal) gene that produces
pale, soft and exudative meat of inferior quality that results to signicant losses in the
meat industry. This study was conducted to detect PSS in pigs from seven farms in Luzon,
Philippines which are used for breeding purposes. They were classied as normal (NN), stress
carrier (Nn) and mutant (nn). This classication will help to form a new breeding system to be
developed ensuring that all offspring are free of the stress gene. Characterization of the Hal
gene was done by collecting blood samples subjected to DNA extraction and genotyping using
mutagenically separated-polymerase chairn reaction (MS-PCR) which is an optimized one step
process of PSS detection. Out of 427 samples, 22 were found to be mutant, 34 were carrier, and
371 were normal. Results for genotypic frequency showed that 87% pigs are normal (NN); 8%
are heterozygotes (Nn) and only 5% are stress-positive (nn). Results were validated through
DNA sequencing which showed the same results with MS-PCR. A genetic screening using this
developed method for the Philippine setting is recommended to be able to minimize the effect
of PSS.
Key words: halothane gene, MS-PCR, pig, porcine stress syndrome
125

MANALAYSAY et al.
Because of the deleterious effects of the
PSS gene, there is a need to manage and
regulate the breeding herd. Manipulation of the
breeding stock will result into a better quality
of pork rendering an increase in its economic
value. By determining the genotypes of the
pigs, it will help breeders to device specic
breeding systems producing PSS gene-free
offspring and ensuring more desirable pork.
Mutagenically Separated-Polymerase
Chain Reaction (MS-PCR) is a PCR-based
technique developed by Rust et al. (1993),
whereby both normal and mutant alleles can
be amplied in the same reaction tube, using
different length allele-specic primers. Thus,
this PCR method does not require the digestion
of an enzyme like PCR-RFLP to determine the
genotypes of the samples, saving more time.
PSS screening using MS-PCR was rst used
by Liaw et al. (2000) in their study with the
Berkshire breed.
The study was conducted to: 1) determine
the PSS genotypes of samples of swine breeding
stock obtained from different farms of Luzon,
Philippines as stress-resistant (NN), stress
carrier (Nn), and stress positive (nn) using
MS-PCR technique; 2) calculate the allelic and
genotypic frequency in the samples of swine
breeding stock and to identify which among
the breed has the highest incidence of PSS
among them; and 3) evaluate the accuracy of
MS-PCR by conrming the results using DNA
sequencing.
This study is limited only in the
determination of the frequencies of the three
genotypes (NN, Nn and nn) of pigs in farms
from Luzon, Philippines. The study does not
cover analysis of quantitative and qualitative
traits of the carcasses of the pigs.
MATERIALS AND METHODS
Blood sample collection
One and a half milliliters of blood samples
were collected from random selection of 427
individual pigs (Pietrain=42, Landrace=109,
Large White=186, Duroc=54 and Chester
White=36) from seven farms in Luzon,
Philippines. The samples were collected
using new and clean syringes to avoid cross
contamination among the pigs and incorrect
genotype identication. The collected blood
samples were stored in tubes treated with
Tris-EDTA buffer to prevent coagulation of the
blood and were transported to the Philippine
Carabao Center Animal Health Laboratory,
Muñoz, Nueva Ecija. Positive controls used
in the study were from the Taiwan Livestock
Research Institute.
DNA extraction
Genomic DNA was isolated from
whole blood samples following a high salt
concentration extraction procedure to facilitate
lysis of erythrocytes and to remove unwanted
debris. White blood cells were concentrated by
adding an approximate volume of ammonium
chloride solution (about 2-3 times the volume
of sample) to the whole blood samples in
microcentrifuge tubes. The solution was mixed
for 15-20 min and centrifuged at 2500 rpm for
5 min. Samples were subjected to successive
washings using ammonium chloride solution
until a white pellet is obtained. Genomic DNA
was extracted using a standard DNA extraction
kit (GF-1 Vivantis, Selangor, Malaysia). DNA
samples were stored at 4°C for the MS-PCR.
MS-PCR
Optimization of PCR was done using
various DNA markers from the halothane
gene with the forward, mutant and reverse
primers (Table 1). These primers were
acquired from Taiwan Livestock Research
Institute, Breeding and Genetics Division.
Prepared DNA samples were amplied using
a DNA polymerase (GoTaq® Flexi, Promega,
Wisconsin, USA). About 20 to 50ng/µl of the
template was added to complete a 10-µl PCR
mixture (SDDH2O, 1x PCR buffer, 3.0 mM
MgCl2, 500 µM deoxynucleoside triphosphates
(dNTPs) and 0.5 µM each primer and 0.05 U/µl
GoTaq® Flexi DNA polymerase). To determine
which annealing temperature is best, Gradient
PCR was conducted. The rst step was 94°C
for 1 min, 65°C for 1 min and 72°C for 1 min in
one cycle, the second step was 94°C for 30 sec,
69°C for 45 sec and 72°C for 45 sec in 35 cycles
followed by a nal extension for 5 min at 72°C.
Representative samples were sent to 1st Base
in Malaysia for DNA sequencing.
126

GENETIC TESTING FOR PORCINE STRESS SYNDROME USING PCR
Gel Electrophoresis and Ultra-Violet
Transillumination
Three and a half grams of agarose and 100
ml of 1X TAE buffer in a 250-ml ask were
heated in a microwave up to its boiling point
with gentle swirling. The resulting mixture
was cooled and 3.5 µg of nucleic acid gel statin
(Gel Red Biotium solution, Biotium, California,
USA) were added per 100 ml gel to allow the
visualization of DNA under the UV light. The
gel bed was sealed and the gel (5-7mm thick)
was allowed to set in UV-transparent gel trays.
The combs were inserted and the gel was
allowed to harden. The combs were removed as
long as the gel nished setting and placed into
an electrophoresis tank.
Addition of 1X TAE buffer to the chamber
was done and the gel was covered to a depth
of approximately 2 mm. Then, a well was
lled with a 5-µl Hyperladder I DNA size
marker. Subsequently, 3-µl of each PCR
samples were loaded into wells beside the
ladder. Electrophoresis was done at 110 V
for 40-60 min and stopped before the dye
runs off at the gel end. After running the
electrophoresis, the resulting gels were placed
in a UV trans-illuminator to view and capture
photographic images and subjected to analysis
and interpretation. The Hal gene gives a
134bp and 114 bp fragments for heterozygous
genotype (Nn), 134bp for mutant allele (nn),
and 114bp for normal allele (NN) under MS-
PCR. The genotypic and allele frequencies of
the three genotypes among the 427 samples
were determined.
RESULTS AND DISCUSSION
In this study, blood samples from 427 pigs
were collected and were subjected to DNA
extraction and genotyping using MS-PCR
wherein 43 of which were boars and 384 were
sows. The DNA of the normal animals had a
size of 114 bp, the carriers had 114 and 134 bp
and the mutant ones have 134 bp (Fig. 1). The
incidences of the genotypes of the 427 animals
were found to be NN=421, Nn=4 and nn=22
(Fig. 2A). The genotypic and allelic frequencies
in the population were also determined (Figs.
2B and 2C). The genotypic frequencies for NN,
Nn and nn were 87%, 8% and 5%, respectively;
while the allelic frequencies for N and n were
91% and 9%, respectively. These frequencies
imply that PSS is present within the swine
population of the Philippines.
Occurrence of PSS can be correlated with
certain breeds since there are specic breeds
that can most likely have PSS gene on them
(Judge et al., 1992). In this study, the highest
incidence of PSS positive was found in Pietrain
(21%), followed by Duroc (13%), Landrace (4%)
and Large White (1%). There was no incidence
of PSS in Chester White breed (Table 2). As
for the PSS carriers, Pietrain had the highest
incidence (29%), followed by Duroc (17%),
Landrace (8%) and Large White (2%). There
was no incidence of PSS in Chester White breed
(Table 3). Furthermore, it was found that boars
had higher incidence of PSS than sows (Table
4).
Representative samples found to be
mutant were sent for DNA sequencing in order
to validate the results. A Cytosine to Thymine
mutation was found on the 49th codon of the
amplied region for the 12 samples sequenced;
this coincides with the results of MS-PCR.
The sequencing also revealed that the region
amplied by the primers using MS-PCR was all
conserved with no polymorphisms, except for
the 49th codon wherein the 1843C-T mutation
was found (Fig. 3). This indicates that the
Table 1. Primers used for optimization of PCR for Porcine Stress Syndrome.
127
1
1
ORIGINAL ARTICLE
Genetic Testing for Porcine Stress Syndrome Using Mutagenically
Separated-Polymerase Chain Reaction
Jessica G. Manalaysay, BS, Domina Flor L. Gamboa, BS, Rubigilda
Paraguison-Alili BS, MS, PhD, Jocelyn G. Rafanan BS, MS
and Claro N. Mingala DVM, MVSc, PhD
Table 1. Primers used for optimization of PCR for Porcine Stress Syndrome.
Primer Name
Primer Sequence (5’3’)*
PCR Product
MS_PSS_CF
CCT GTG TGT GTG CAA TGG TGT GGC
CGT CC
Normal (NN) – 114 bp
MS_PSS_TF
GTG CTG GAT GTC CTG TGT TCA ATG
TGT GTG TGC AAT GGT GTG GCC GGC T
Carrier (Nn) – 114 and 134
bp
MS_PSS_Rev
CTG GTG ACA TAG TTG ATG AGG TTT
GTC TGC
Mutant (nn) – 134 bp
Table 2. Incidence of Porcine Stress Syndrome positive among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
9
21%
Landrace
109
4
4%
Large White
186
2
1%
Duroc
54
7
13%
Chester White
36
0
0%
Table 3. Incidence of Porcine Stress Syndrome carriers among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
12
29%
Landrace
109
9
8%
Large White
186
4
2%
Duroc
54
9
17%
Chester White
36
0
0%
Table 4. Incidence of Porcine Stress Syndrome positive and carrier with regards to sex.
Animal
Sample #
PSS
positive
Incidence
(PSS positive)
PSS
carrier
Incidence
(PSS carrier)
Sows
384
16
4.17%
32
8.33%
Boar
43
6
13.85%
2
4.65%

MANALAYSAY et al.
primer used is very specic.
From the results stated above, it can be
said that MS-PCR could be used to detect
PSS not only because of its accuracy but also
because of its time efciency. The present
methods being used to screen PSS are the
Polymerase Chain Reaction Restriction
Fragment Length Polymorphism (PCR-RFLP)
and Real Time Polymerase Chain Reaction
(RT-PCR). PCR-RFLP is a time-consuming
method for it requires a restriction enzyme that
needs a digestion period before the genotype
can be determined. The enzyme takes at least
2 h (Jin et al., 2005) before it can digest the
PCR product. As for RT-PCR, though a very
sensitive and accurate method, the probes and
128
Fig. 1. Agarose gel electrophoresis of MS-PCR test for the Hal gene. Lane 1 and 7- 50bp molecular weight
ladder. Lanes 2-4 and 8-10 are classied as normal (NN); Lane 5 is classied as mutant (nn); Lanes 6,
11, and 12 are carrier (Nn).
Fig. 2. Graphs showing the incidence and frequencies of the Hal gene. A) The incidence of the three
genotypes in a population of 427 pigs (NN=371, Nn=34 and nn=22). B) Genotypic frequency for PSS
of the 427 pigs (NN=87%, Nn=8% and nn=5%). C) Allelic frequency of the Hal gene in a population of
427 pigs (N=91% and n=9%).

GENETIC TESTING FOR PORCINE STRESS SYNDROME USING PCR
dyes needed are very expensive.
Manipulation of the breeding system can
be done via molecular analysis such as genetic
screening using live pigs which is a fast and
noninvasive type of testing (Bastos et al.,
2000). By detecting the genotypes of the sows
and boars, breeders may be able to design
specic breeding systems of their interest.
Although it has been found that the n allele
may give positive effects, these were offset by
its negative effects which are high drip loss,
soft and pale color (Jin et al., 2005) reecting
the inability of the meat to undergo further
processing in the meat industry (Du, 2004).
However, breeders have the liberty to decide if
they will eliminate the n allele or utilize it in
relation to the improvement of meat production
qualitatively or quantitatively.
The results of the study showed that
MS-PCR is an accurate method that can be
used to screen for Porcine Stress Syndrome.
Elimination of the mutated gene is required to
prevent the prevalence of its negative effects
to the next generation. The study recommends
that the method be used as standard protocol
for screening of PSS in the Philippines in order
to reduce the frequency of PSS.
ACKNOWLEDGMENT
The authors thank Dr. Arnel N. Del Barrio,
Philippine Carabao Center (PCC) Executive
Director, for the use of the laboratory facilities,
Table 2. Incidence of Porcine Stress Syndrome positive among different breeds.
129
Table 3. Incidence of Porcine Stress Syndrome carriers among different breeds.
Table 4. Incidence of Porcine Stress Syndrome positive and carrier with regards to sex.
1
1
ORIGINAL ARTICLE
Genetic Testing for Porcine Stress Syndrome Using Mutagenically
Separated-Polymerase Chain Reaction
Jessica G. Manalaysay, BS, Domina Flor L. Gamboa, BS, Rubigilda
Paraguison-Alili BS, MS, PhD, Jocelyn G. Rafanan BS, MS
and Claro N. Mingala DVM, MVSc, PhD
Table 1. Primers used for optimization of PCR for Porcine Stress Syndrome.
Primer Name
Primer Sequence (5’3’)*
PCR Product
MS_PSS_CF
CCT GTG TGT GTG CAA TGG TGT GGC
CGT CC
Normal (NN) – 114 bp
MS_PSS_TF
GTG CTG GAT GTC CTG TGT TCA ATG
TGT GTG TGC AAT GGT GTG GCC GGC T
Carrier (Nn) – 114 and 134
bp
MS_PSS_Rev
CTG GTG ACA TAG TTG ATG AGG TTT
GTC TGC
Mutant (nn) – 134 bp
Table 2. Incidence of Porcine Stress Syndrome positive among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
9
21%
Landrace
109
4
4%
Large White
186
2
1%
Duroc
54
7
13%
Chester White
36
0
0%
Table 3. Incidence of Porcine Stress Syndrome carriers among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
12
29%
Landrace
109
9
8%
Large White
186
4
2%
Duroc
54
9
17%
Chester White
36
0
0%
Table 4. Incidence of Porcine Stress Syndrome positive and carrier with regards to sex.
Animal
Sample #
PSS
positive
Incidence
(PSS positive)
PSS
carrier
Incidence
(PSS carrier)
Sows
384
16
4.17%
32
8.33%
Boar
43
6
13.85%
2
4.65%
1
1
ORIGINAL ARTICLE
Genetic Testing for Porcine Stress Syndrome Using Mutagenically
Separated-Polymerase Chain Reaction
Jessica G. Manalaysay, BS, Domina Flor L. Gamboa, BS, Rubigilda
Paraguison-Alili BS, MS, PhD, Jocelyn G. Rafanan BS, MS
and Claro N. Mingala DVM, MVSc, PhD
Table 1. Primers used for optimization of PCR for Porcine Stress Syndrome.
Primer Name
Primer Sequence (5’3’)*
PCR Product
MS_PSS_CF
CCT GTG TGT GTG CAA TGG TGT GGC
CGT CC
Normal (NN) – 114 bp
MS_PSS_TF
GTG CTG GAT GTC CTG TGT TCA ATG
TGT GTG TGC AAT GGT GTG GCC GGC T
Carrier (Nn) – 114 and 134
bp
MS_PSS_Rev
CTG GTG ACA TAG TTG ATG AGG TTT
GTC TGC
Mutant (nn) – 134 bp
Table 2. Incidence of Porcine Stress Syndrome positive among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
9
21%
Landrace
109
4
4%
Large White
186
2
1%
Duroc
54
7
13%
Chester White
36
0
0%
Table 3. Incidence of Porcine Stress Syndrome carriers among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
12
29%
Landrace
109
9
8%
Large White
186
4
2%
Duroc
54
9
17%
Chester White
36
0
0%
Table 4. Incidence of Porcine Stress Syndrome positive and carrier with regards to sex.
Animal
Sample #
PSS
positive
Incidence
(PSS positive)
PSS
carrier
Incidence
(PSS carrier)
Sows
384
16
4.17%
32
8.33%
Boar
43
6
13.85%
2
4.65%
1
1
ORIGINAL ARTICLE
Genetic Testing for Porcine Stress Syndrome Using Mutagenically
Separated-Polymerase Chain Reaction
Jessica G. Manalaysay, BS, Domina Flor L. Gamboa, BS, Rubigilda
Paraguison-Alili BS, MS, PhD, Jocelyn G. Rafanan BS, MS
and Claro N. Mingala DVM, MVSc, PhD
Table 1. Primers used for optimization of PCR for Porcine Stress Syndrome.
Primer Name
Primer Sequence (5’3’)*
PCR Product
MS_PSS_CF
CCT GTG TGT GTG CAA TGG TGT GGC
CGT CC
Normal (NN) – 114 bp
MS_PSS_TF
GTG CTG GAT GTC CTG TGT TCA ATG
TGT GTG TGC AAT GGT GTG GCC GGC T
Carrier (Nn) – 114 and 134
bp
MS_PSS_Rev
CTG GTG ACA TAG TTG ATG AGG TTT
GTC TGC
Mutant (nn) – 134 bp
Table 2. Incidence of Porcine Stress Syndrome positive among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
9
21%
Landrace
109
4
4%
Large White
186
2
1%
Duroc
54
7
13%
Chester White
36
0
0%
Table 3. Incidence of Porcine Stress Syndrome carriers among different breeds.
Breed
Sample #
PSS positive
Incidence
Pietrain
42
12
29%
Landrace
109
9
8%
Large White
186
4
2%
Duroc
54
9
17%
Chester White
36
0
0%
Table 4. Incidence of Porcine Stress Syndrome positive and carrier with regards to sex.
Animal
Sample #
PSS
positive
Incidence
(PSS positive)
PSS
carrier
Incidence
(PSS carrier)
Sows
384
16
4.17%
32
8.33%
Boar
43
6
13.85%
2
4.65%

MANALAYSAY et al.
Jin H, Park B, Park J, Hwang I, Lee S, Yeon S, Kim
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130
the Department of Science and Technology-
Philippine Council for Agriculture, Aquatic and
Natural Resources Research and Development
for the research grant under the Swine
Genomics Project 2 and the staff of the Animal
Health Unit of PCC for their technical support.
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Fig. 3. DNA sequence analysis of the Hal gene. The sequence of the samples was aligned and compared
with the sequence in the GenBank (Accession number: XM_005664538.1). Nucleotide mutation (C/T)
was observed in 49th loci of the amplied region.