Prediction of the halothane (Hal) genotypes of pigs by deducing Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in Swedish breeds

Results from a large-scale study, comprising 75 different breeding herds, are reported on predicting the halothane (Hal) genotypes of individual pigs by making use of the known close linkage between Hal and three electrophoretic blood marker loci (Phi, Po2, Pgd). The parents haplotypes (involving Hal and marker loci) were determined from the HAL phenotypes (halothane test results) and marker loci phenotypes of their offspring in the first one or two litters studied. In subsequent litters of the Hal-marker loci haplotyped parents, the offspring's expected Hal genotypes could be predicted on the basis of the marker loci haplotypes inherited by them. By comparing the expected and observed HAL phenotypes of offspring in subsequent litters, the predicted Hal genotype was found to be correct in 90-95% of the 4000 offspring (from Nn X Nn and Nn X nn matings) of Swedish Landrace and Yorkshire breeds studied. The order of the three marker loci was confirmed as Phi-Po2-Pgd but the position of Hal with regards to Phi could not be resolved. The recombination frequencies between the most distant loci in this region, viz. Hal-Pgd and Phi-Pgd, were estimated to be 3-4.5% and 4-6%, respectively. The easy and rapid electrophoretic techniques described in the study to phenotype PHI, PO2, PGD, also allowed phenotyping of six other polymorphic protein systems on the same gels. Thus Hal genotyping and effective parentage control can be conducted simultaneously.     

Gene order and recombination rates in the linkage group S-Phi-Hal-H-(Po2)-Pgd in pigs

Families of Czech Landrace (94 litters and 636 offspring) were tested for halothane sensitivity, A-O (S), H, PHI and PGD phenotypes. Informative matings for the estimation of recombination rates between marker loci were selected. The following recombination frequencies were established: S - Phi = 4.8% (2.5%-10.7%); S - H = 6.8% (4.3%-11.7%); Phi - H = 2.6% (0.9%-5.3%); H - Pgd = 4.4% (1.6%-8.0%). Cross-overs were observed also between S - Hal, Hal - H and Hal - Pgd, but were not found between Phi - Hal. On the basis of these results it has been possible to revise the position of the S locus in this linkage group. The most probable gene order would be: S - Phi - Hal (or Hal - Phi) - H - (Po2) - Pgd. A striking difference was found between the number of halothane-sensitive pigs (87) and HalnHaln genotypes determined by haplotyping (123). Segregation rates in 19 backcross matings and experimental matings of the animals proved that this difference is mostly due to incomplete penetrance or low expression of halothane sensitivity.     

Gene order and recombination rates in the linkage group S-Phi-Hal-H-(Po2)-Pgd in pigs

Families of Czech Landrace (94 litters and 636 offspring) were tested for halothane sensitivity, A-O (S), H, PHI and PGD phenotypes. Informative matings for the estimation of recombination rates between marker loci were selected. The following recombination frequencies were established: S-Phi = 4.8 % (2.5 % -10.7 %);S-H = 6.8 % (4.3 %-11.7 %); Phi-H = 2.6 % (0.9 %-5.3 9%); H-Pgd = 4.4 % (1.6 %-8.0 %). CCCC-overs were observed also between S- Hal, Hal-H andHal - Pgd, but were not found between Phi - Hal. On the basis of these results it has been possible to revise the position of the S locus in this linkage group. The most probable gene order would be: S - Phi - Hal (or Hal - Phi) -H- (P02) - Pgd.
A striking difference was found between the number of halothane-sensitive pigs (87) and HalⁿHal ⁿ genotypes determined by haplotyping (123). Segregation rates in 19 backcross matings and experimental matings of the animals proved that this difference is mostly due to incomplete CCC or low expression of halothane sensitivity.     

Blood group association with severity and speed of the halothane reaction

The second generation (n = 227) of British Landrace pigs from selected halothane-positive parents (36 litters) were blood-typed for the S(A-O), H and Phi loci and subjected to four 5-minute halothane tests at 21, 35, 49 and 63 days of age. Cumulative scores based on severity and speed of reaction were analysed in relation to single-locus blood group genotypes and linkage group sequences at two and three loci. A highly significant negative correlation (r = -0.79) was found between severity and speed of reaction. Significant differences occurred between blood group genotypes and linkage groups in both severity and speed of reaction. Genotypes S s/s, H a/a or H a/- and Phi B/B and linkage groups involving these three types had the highest cumulative reaction score and the fastest reaction time, whereas genotypes Phi A/B, S S/S or S S/s and H a/cd and linkage groups with these types had the lowest and slowest reaction scores. Some differences between genotypes and linkage groups were attributed to phenotypically halothane-positive parents and offspring being genotypically Hal N/n. These effects could result from linkage with heterozygous types such as H a/cd and S S/s. The possible role of the H cd allele acting as a genetic marker for a suppressor gene to the halothane reaction is discussed.     

Linkage studies between the halothane (Hal), phosphohexose isomerase (Phi) and the S(A -O) and H red blood cell loci of Pietrain/ Hampshire and Landrace pigs

Frequency of blood group factors at the A-O and H loci were markedly altered within halothane positive (HP) and halothane negative (HN) composite synthetic Pietrain/Hampshire lines (PTH) over four generations of selection.
Linkage studies on the litters from 45 double backcross and 20 mixed and intercross matings, involving the S(A-O), H, Phi and Hal loci, were made in the PTH line and halothane positive and negative selected British Landrace lines. Crossing-over frequencies of 0.05 ± 0.04, 0.05 ± 0.03 and 0.1 ± 0.03 were established between Phi and Hal, H and Hal , and Phi and H respectively. An unequal crossing-over frequency between Phi and H was found when the alleles Ha and Hcd were compared. The difference in recombination frequency between the Ha and Hcd alleles amounted to 0.04 to 0.06.
No cross-overs were observed between the S(A -O ) and Phi, H or Hal loci in 15 families studied. The position of the S locus in relation to the other loci could not be established, but statistical evidence of association favours a haplotype sequence of Phi-Hal-S-H .     

Blood group association with severity and speed of the halothane reaction1

The second generation (n= 227) of British Landrace pigs from selected halothane-positive parents (36 litters) were blood-typed for the S(A-0), H and Phi loci and subjected to four 5-minute halothane tests at 21, 35, 49 and 63 days of age. Cumulative scores based on seventy and speed of reaction were analysed in relation to single-locus blood group genotypes and linkage group sequences at two and three loci. A highly significant negative correlation (r = -0.79) was found between severity and speed of reaction. Significant differences occurred between blood group genotypes and linkage groups in both severity and speed of reaction. Genotypes S s/s, H a/a or H a/- and Phi B/B and linkage groups involving these three types had the highest cumulative reaction score and the fastest reaction time, whereas genotypes Phi A/B, S S/S or S S/s and H a/cd and linkage groups with these types had the Iowest and slowest reaction scores. Some differences between genotypes and linkage groups were attributed to phenotypically halothane-positive parents and offspring being genotypically Hal N/n. These effects could result from linkage with heterozygous types such as H a/cd and S S/s. The possible role of the H ^cd allele acting as a genetic marker for a suppressor gene to the halothane reaction is discussed.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electrophoresis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles (Po-2F and Po-2s) at a single locus. The frequency of Po-2F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phosphohexose isomerase locus (Phi). A recombination frequency of 3.2% was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven recombinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd.     

Genetic variation at a pig serum protein locus, Po-2 and its assignment to the Phi, Hal, S, H, Pgd linkage group

Two-dimensional agarose gel (pH 5.4)-polyacrylamide gel (pH 9.0) electropho-resis of pig serum samples revealed a new serum protein (postalbumin-2, PO-2) polymorphism. Family data supported the hypothesis that the three PO-2 phenotypes observed were controlled by two codominant, autosomal alleles ( Po-2^F and Po-2^S ) at a single locus. The frequency of Po-2^F in Swedish Landrace and in Swedish Yorkshire breeds was estimated at 0.74 and 0.65, respectively. Evidence was presented for close genetic linkage between Po-2 and the red cell phospho-hexose isomerase locus ( Phi ). A recombination frequency of 3.2 % was obtained from double backcross material. Data obtained in a Danish Landrace material showing linkage between the Po-2 locus and the H blood group locus, the Pgd locus and Hal (locus for halothane sensitivity) are also given. A total of seven re-combinants were observed. They show that Po-2 is a new locus in a previously established linkage group. The likely sequence of the loci is: Phi, Hal, S, H, Po-2, Pgd .     

Associations between marker genotypes, halothane reaction, creatine kinase activity and meat quality characters in a sample of German Landrace pigs

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H. The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles PhiA (0.48), H- (0.43) and PgdA (0.70) gave quite different results. The frequency of halothane-positive pigs found by using a severe test was only 30%. Only 5.4%, 8.8%, 13.4% and 13.9% of animals with PhiA/A, H-/-, PgdA/A and PhiA/B genotypes respecitively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative (N/N) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd, with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H-/-. In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.(ABSTRACT TRUNCATED AT 250 WORDS)     

Associations between marker genotypes, halothane reaction, CC activity and meat quality characters in a sample of German Landrace pigs

Routine blood typing of German Landrace pedigree populations and an earlier study revealed very low frequencies of the favourable alleles at the marker loci Phi, Pgd and H . The hypothesis was that in this population the whole linkage group of favourable alleles at the halothane and neighbouring marker loci may have been lost as a consequence of intense selection for leanness and type. The present study of 1050 German Landrace pigs at the Relliehausen experimental station, where some effort has been made to maintain a higher frequency of the favourable alleles Phi^A (0.48), H- (0.43) and Pgd^A (0.70) gave quite different results.
The frequency of halothane-positive pigs found by using a severe test was only 30 %. Only 5.4 %, 8.8 %, 13.4 % and 13.9 7% of animals with Phi^AIA, H^-I-, Pgd^A/A and Phi^A/B genotypes respectively were halothane-positive. Forty to sixty per cent of pigs with these marker genotypes could therefore be expected to be homozygous halothane-negative ( N/N ) animals. Creatine kinase activity and three selected meat quality characters showed highly significant differences between the A/A and the B/B genotypes for the marker loci Phi and Pgd , with the heterozygotes being intermediate. These differences are greater than those observed between halothane-negative and halothane-positive phenotypes. The only other consistently superior marker genotype in this population was the H blood group genotype H -^I- . In contrast to findings from Sweden and Switzerland, the postalbumin locus Po2 and the suppressor locus S for the A-O blood groups did not exhibit useful marker qualities.
It is concluded that in the German Landrace the marker loci Phi, Pgd and H could also be helpful in breeding homozygous halothane-negative pigs with distinctly better meat quality characteristics.     

Influence of the halothane gene (HAL) on pork quality in two commercial crossbreeds

We evaluated the effect of the halothane (HAL) gene on the quality of pork in domestic pigs. Half-carcasses from two different commercial pig (Sus domestica) crossbreeds were analyzed, 46 of which were homozygous dominant (HAL(NN)) and 69 of which were heterozygous (HAL(Nn)) for the halothane gene. The measures included backfat thickness, lean meat percentage, carcass weight, pH 24 h after slaughtering, color, and drip loss; DNA was extracted from the haunch muscle. Swine with the HAL(Nn) genotype had less backfat thickness and higher lean meat percentages than swine with the HAL(NN) genotype. Yet, swine with the HAL(Nn) genotype had lower quality meat than those with the HAL(NN) swine. The pH at 24 h was lower in HAL(Nn) swine. The meat color was paler in HAL(Nn) animals, the drip loss was greater in those animals bearing the n allele, and the amount of intramuscular fat was not related to the halothane genotype. We conclude that bearers of the recessive allele of the halothane gene produce more meat, but with quality parameters that are inferior to those sought by consumers and industry.     

Localization of the Po2 locus in the S, Phi, Hal, H, Po2, Pgd linkage group in pigs

The localization of the Po2 locus controlling a polymorphic serum postalbumin was studied in 41 families of the Czech Landrace breed. The haplotypes involving six closely linked loci (S, Phi, Hal, H, Po2, Pgd) were determined for each family member. The crossovers observed between the H, Po2 and Pgd loci indicated that Po2 is located between H and Pgd. The Po2 locus appears to be closer to H [theta = 0.54% (0.06%-1.92%)] than to Pgd [theta = 4.02% (1.67%-7.96%)]. A strong Ha-Po2S association (r = 0.96, P less than 0.001) and H-PO2 linkage disequilibrium (D = 0.2218, P less than 0.01, D/DMax = 0.98) were found.     

Linkage of genes for PHI,halothane sensitivity,A-O inhibition,H red blood cell antigens and 6-PGD variants in pigs*

Data from one apparent crossover between S and H, two between PHI and HAL on one side and S on the other, and one between PHI on one side and HAL, S and H on the other, indicate a gene order in pigs of Phi-Hal-S-H-Pgd for genes for PHI, halothane sensitivity, inhibition of expression of A and O, H red blood cell antigens and 6-PGD types. Rasmusen et al. (1980) provided data for a gene order in pigs ofPhi-Hal-H-Pgd for genes for phosphohexose isomerase (PHI) isozyme variants, halothane sensitivity (HAL), H red cell antigens and 6-phosphogluconate dehydrogenase (6-PGD) variants, and suggested that there might be a locus for a gene for inhibition of expression of A and O separate from the locus for H. This is contrary to an earlier proposal by Rasmusen (1972) that the H-system genotype directly influences expression of A and O. Imlah (1980) suggested that the recessive gene for halothane sensitivity has a suppressant effect on the expression of A and O. Andresen (1981) proposed that the locus for inhibition of A and O (for which Rasmusen, 1964, proposed the symbol S) was between the loci for HAL and H types. Data presented in Table 1, which includes haplotypes for three recombinant offspring described by Rasmusen et al. (1980) (883-1, 233-3 and 3864-1) as well as one other recombinant (296-2) provide evidence for the gene order for five genes proposed by Andresen. Types for 6-PGD are listed for all pigs, although they do not provide evidence for gene order in these cases. Male 883-1 (Table 1, and Rasmusen et al., 1980, Table 5) provided the original evidence for recombination between S and H. His phenotype, as well as his genotype as revealed by progeny test (Rasmusen et al., 1980, Table 6) indicated that recombination had occurred between the genes for PHI, HAL and S and the gene for H type in his dam, so that the S locus mapped between H and the loci for the other three traits. The phenotype of one of his sons (233-3, Table 1, and Rasmusen et al., 1980, Table 6) indicated that there had been a recombination between genes for PHI and HAL types on one side and S and H types on the other, providing evidence that the S locus was separate from PHI and HAL as well as H. Another pig listed in Table 1,3864-1, was also described by Rasmusen et al. (1980, Table 9) as a recombinant. This pig provides evidence for recombination between PHI on one side and HAL, S and H on the other, establishing a gene order of Phi-Hal-S-H-Pgd. The last pig listed in Table 1,296-2, is a recombinant comparable to 233-3. The H type of his dam provides markers indicating the recombination was between PHI and HAL on one side and S and H on the other, although the unusual expression of HAL phenotype in both parents of 296-2 makes her haplotypes somewhat uncertain. (Recombination may have been between PHI and HAL rather than as indicated in Table 1.) In spite of incomplete penetrance for HAL (Ollivier et al., 1975; Smith & Bampton, 1977) which makes haplotypes for HAL questionable in some cases, the other genetic markers available are useful to show that recombination has taken place. Without considering the results of halothane testing, if the apparent recombinants are accepted as being as indicated, the order of the genes at the other four loci seems established. Alleles for S types appear to be separable by recombination from those for PHI and H, and the S locus appears to be between the loci for PHI and H. For the five loci, data obtained thus far are cohsistent with a gene order of Phi-Hal-S-H-Pgd.     

The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi . Therefore the gene sequence S - Phi - Hal -H- Po2 -Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.     

The position of the epistatic S locus in the halothane linkage group in pigs

The linkage of the Phi, Pgd, Po2, S, H and halothane sensitivity loci was followed in a Belgian Landrace family, heterozygous for these systems over 6 generations. Recombination next to the S locus occurred mainly in pigs belonging to this particular family. From this investigation the position of the S locus is proved to be outwith the Phi-Pgd region, next to Phi. Therefore the gene sequence S - Phi - Hal - H - Po2 - Pgd is proposed. Higher recombination rates were observed in the female parental line of the multiheterozygous family when compared to the male parental line. Additional data from animals, unrelated to this strain, confirm the evidence of close linkage of the S system to the nearest marker loci.     

Recombination between the S and the Hblood group loci in Pigs

The investigation of A-O blood group phenotypes in selected pig families confirms the existence of an S locus which specifically controls the expression of A and) alloantigens. Forty-five informative litters were scored for linkage between the S gene and the H blood group locus. The recombination frequency in 345 offspring was estimated to be 9.56 % (33 cross-overs). Specific difficulties involved in the determination of A-O and H phenotypes and the importance of S and H polymorphisms for the determination of Hal genotypes are discussed.     

Hsp70 response in pigs is affected by their Halothane genotypes after heat stress

 Heat shock protein ([Hsp70]) and ionized calcium ([Ca²⁺]) concentrations were examined in pigs’ blood at different times after heat stress (HS). Pigs were from different halothane genotypes (HAL) (NN, n=7; Nn, n=5; nn, n=6). [Hsp70] increased significantly from 1–2 h after HS and [Ca²⁺] decreased significantly from HS-end in NN and Nn. No significant differences in [Hsp70] and [Ca²⁺] were detected among NN and Nn. [Hsp70] and [Ca²⁺] in nn did not vary significantly from baseline values. [Hsp70] was significantly higher in NN and Nn than in nn. Briefly, HAL affected [Hsp70] after HS in pigs probably via a disordered [Ca²⁺] regulation.     

A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for high-affinity potassium uptake even in the absence of Na⁺ stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt −195/−189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K⁺-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.     

Polymorphic plasma postalbumins of some domestic animals (pig PO2, horse Xk and dog Pa proteins) identified as homologous to human plasma α1B-glycoprotein

Pig, horse and dog plasma proteins, separated by horizontal polyacrylamide gel electrophoresis (pH 9.0) and electrophoretically transferred to nitrocellulose membranes, were tested for cross-reaction with antiserum to human plasma alpha 1B-glycoprotein (alpha 1B). The results showed that one previously reported polymorphic plasma postalbumin in each of these species (pig PO2, horse Xk and dog Pa protein) was homologous to human plasma alpha 1B. In the light of the previously known genetic linkages in these species, this implied: (1) alpha 1B gene is close linked to Phi, Pgd and Hal (halothane sensitivity locus) loci in pigs; and (2) alpha 1B gene is linked to ME1 and Phi loci in horses. This suggested that the alpha 1B gene may also be found to be closely linked to gene(s) controlling susceptibility to malignant hyperthermia in humans and other mammals.