Characterization of swine stress gene by DNA testing using plucked hair as a source of DNA

O gene do estresse suino (hal) em homozigose recessiva (nn) ocasiona a sindrome do estresse porcino (PSS), e esta relacionado com a ocorrencia da carne palida, mole e exudativa (PSE). Em heterozigose (Nn) esta associado com diminuicao da qualidade de carcaca. Um total de 179 suinos (86 Large White, 69 Landrace, 12 Duroc e 12 Pietrain) foram caracterizados como homozigotos normais (NN), heterozigotos e homozigotos recessivos por analise do DNA usando a reacao de polimerizacao em cadeia (PCR), seguida de um ensaio com endonuclease de restricao. Foi utilizado foliculo piloso como fonte de DNA genomico. O produto do PCR foi digerido com a enzima de restricao CfoI, seguindo-se analise dos fragmentos por eletroforese em gel de agarose. Dentre os 179 animais analisados, 126 (70,0%) foram caracterizados como NN, 50 (28,0%) como Nn, e 3 (2,0%) caracterizados como nn. A frequencia de heterozigotos foi maior (P < 0,05) em animais da raca Landrace do que em animais da raca Large White. Nove animais da raca Pietrain foram classificados como heterozigotos e tres como homozigotos recessivos. Estes resultados podem estar relacionados com a incidencia de PSS e PSE nestas racas, as quais sao largamente utilizadas em programas de cruzamento. A utilizacao de foliculo piloso como fonte de DNA genomico foi um metodo rapido e nao-invasivo que viabiliza a execucao da caracterizacao do gene hal.


INTRODUCTION
The swine stress gene (hal) is located on chromosome 6 (p1.1-q2.1)and codes for ryanodine receptors, which are Ca 2+ release channels of skeletal muscle sarcoplasmic reticulum (Otsu et al., 1991).Porcine stress syndrome (PSS) is an inherited myopathology in which skeletal muscle contraction, hypermetabolism and an elevation in body temperature are triggered by inhaled anesthetics and environmental stress (Louis et al., 1990;Santoro and Faucitano, 1996).Comparison of sequence of full-length hal cDNA (Genbank M91451) from PSS susceptible and PSS non-susceptible pigs has revealed 18 single nucleotide polymorphisms between these two types of pigs.One of the polymorphisms involves the substitution of cytosine (PSS non-susceptible) by a thymine (PSS susceptible) at nucleotide 1843.This alteration results in the replacement of an arginine at position 615 by a cysteine (Fujii et al., 1991).As a consequence, in recessive homozygotes (nn) the gene hal leads to PSS and the major post-mortem manifestation of pale, soft and exudative pork (PSE).In heterozygosis (Nn) the hal gene produces lower carcass quality (Prommier and Houde, 1993;Cheak et al., 1994;Webb, 1996), but possibly higher carcass weight (Zhang et al., 1992;Leach et al., 1996;Fávero, 1997).The polymorphism at nucleotide 1843 of the hal gene has recently been characterized by a DNA test using blood or a muscle biopsy as the source of genomic DNA (Houde et al., 1993;Cheak et al., 1994).
In this study, we assessed the validity of the DNA test in commercial pigs using PCR amplification of the target region of hal gene followed by a restriction endonuclease assay (REA).We also examined the feasibility of using plucked hair as source of genomic DNA, and determined the frequency of each hal genotype in Landrace, Large White, Duroc and Pietrain pig breeds.

Animals
One hundred seventy-nine pigs (86 Large White, 69 Landrace, 12 Duroc and 12 Pietrain) were studied.All animals came from a single commercial farm in the State of Rio Grande do Sul, Brazil.Plucked hair was collected from randomly chosen pigs.

Extraction of genomic DNA
Genomic DNA was extracted from the hair roots using two protocols.In protocol I (Bauerová et al., 1995), 10 hair roots were suspended in 100 µl of lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 2% SDS, 70 mM DTT and 0.4 mg/ml of proteinase K), and then incubated at 45°C overnight.Five microliters was used in the PCR reaction.In protocol II, modified from Drissing et al. (1996), 1-3 hair roots were incubated at room temperature for 5 min in 0.5 µl of 0.1 M NaOH, after which 4.5 µl of 0.02 M Tris-HCl, pH 7.4, was added.The PCR reaction mixture (20 µl) was then added to the tube containing the above extract.As alternative sources of DNA, blood and semen samples were also used and the DNA was extracted according to the method described by Sambrook et al. (1989).

PCR and REA
The PCR reactions were done in a final volume of 25 µl containing 200 µM of each dNTP, 2.5 µl of PCR buffer with a final concentration of 1.5 mM MgCl 2 , 0.2 µM of each primer (forward 5'-GTTCCCTGTGTGGCAATGGTG-3' and reverse 5'-ATCTCTAGAGCCAGGGAGCAAGTTCT CAGTAAT-3') (Fujii et al., 1991), 1 U of Taq DNA polymerase (CENBIOT/RS, UFRGS) and 5 µl of extraction solution containing genomic DNA (Innis et al., 1990).The PCR reaction was carried out in a Perkin Elmer 2400 thermocycler with the following settings: 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C for 30 cycles with a final cycle of 7 min at 72°C.Five microliters of the PCR product was digested with 5 U of CfoI (Boehringer Mannhein) for 3 h at 37°C.Digestion by CfoI produced two DNA fragments of 49 pb and 32 pb for the N allele and an intact 81 pb for the "n" allele.The digested product was submitted to electrophoresis on a 4% agarose gel containing 0.5 µg of ethidium bromide/ml (Sambrook et al., 1989), visualized with an UV transilluminator and photographed using a Kodak DC40 digital camera.

Gene frequency
The genotype frequencies of the hal gene (NN, Nn, and nn) among the breeds were analyzed using the chi-square test (χ 2 ).

RESULTS AND DISCUSSION
PCR amplified a specific 81-base pair (bp) DNA fragment from the hal gene using different sources of pig DNA.Collection and transportation of plucked hair were quite easy so this was the source used throughout this study.Protocol II for DNA extraction from plucked hair gave the most consistent results, and since it was simpler and easier to perform than protocol I, this method was adopted as standard.A single hair root was enough to allow amplification of the expected DNA fragment (Figure 1).The impurities present in the DNA samples apparently did not affect amplification of the target DNA.The size of the fragment amplified by PCR was the same, 81 bp, in all samples analyzed.
Digestion of the PCR product with CfoI produced two fragments of 49 bp and 32 bp for normal homozygotes (NN), three fragments of 81 bp, 49 bp and 32 bp for heterozygotes (Nn) and an undigested fragment of 81 bp for recessive homozygotes (nn) (Figure 2).
The gene frequency of the n allele of the hal gene was higher (P < 0.05) in Landrace than in Large White animals (Table I).The frequency of heterozygotes was higher (P < 0.05) in Landrace pigs than Large White pigs.
Comparisons between Duroc and Pietrain pigs were not done, because of the small sample size.However, there was a tendency towards a high frequency of the n allele in   Plucked hair was a suitable source of DNA for PCR, and it was easily collected and transported from the farm to the laboratory.This source of DNA should facilitate the characterization of the hal genotype in farm animals.Collection and use of blood or semen as a source of DNA is 1 2 3 4 5 6 7 8 9 10 11 bp 100 050 010 laborious and, in some cases, dangerous because of the stress caused to the animal during sample collection.Death may occur if the genotype for the hal gene is nn.
The high frequency of heterozygotes (Nn) in Landrace pigs and the high frequency of heterozygotes (Nn) and recessive homozygotes (nn) in Pietrain pigs may be related to the incidence of PSS and PSE, since both of these breeds are used widely in breeding programs and commercial farms.A study to correlate the presence of the n allele with carcass traits in these animals was done by Bastos et al. (2000), and no correlation was found between the presence of the n allele and higher carcass weight, contrary to findings of Zhang et al. (1992), Leach et al. (1996) and Fávero (1997).
As the presence of the hal gene, both in heterozygosity and recessive homozygosity, does not seem to be associated with better carcass weight, and the utilization of these animals may lead to lower carcass quality, breeding programs should aim at eliminating this genotype from the herd.The use of hair roots as a source of DNA should facilitate the analysis of the genotype of hal gene of different breeds and crossbreed pigs.

Figure 1 -
Figure 1 -PCR products obtained using DNA extracted with protocol II.Lane 1: 100-bp ladder molecular weight marker (Gibco BRL); lane 2: DNA extracted from a single hair root; lane 3, DNA extracted from two hair roots, and lane 4: DNA extracted from three hair roots.
a low frequency of this allele in Duroc pigs.No Pietrain animal was characterized as NN.