Position of the Phi and Po2 loci in the Hal linkage group in pigs

Carte genetique des loci Phi et Po2 dans le groupe de linkage Hal chez le porc etudie par caracterisation du phenotype de 165 portees et de 1348 descendants pour la sensibilite a l'halothane et pour les alleles de A-O(S), 4, Phi, PGD et Po2


Introduction
The linkage between the H blood group locus and the loci for the variants of 6-phosphogluconate dehydrogenase (Pgtn and phosphohexose isomerase (Phi) was first described by Andresen (1971 ). Rasmusen & Christian (1976) reported an association between H genotypes and susceptibility to halothane-induced stress. Jorgensen et al. (1976) postulated that the association between H and porcine stress and the linkage of Phi and H was the causal link for the association between Phi genotypes and stress susceptibility.
The inheritance of halothane-induced stress has been shown to be controlled by a recessive gene at a single locus (Han with incomplete penetrance (Ollivieret al., 1975;Smith & Bampton, 1977). N symbolizes Hal N and n, Hal n. Pigs that are N/N or Nln should therefore be HAL-non reactors and nln signifies a HAL + reactor.
Linkage studies between Hal and Phi have not made it possible to place the Hal locus accurately within the linkage group. Using a method for calculation of relative linkage disequilibrium coefficients, Andresen (1979) proposed that Halwas located between Phi and H. The gene order Phi-Hal-H-Pgd was also supported by Rasmusen et al. (1980). Their data, however, did not permit them to distinguish between the order Phi-Hal-H-Pgd as opposed to Hal-Phi-H-Pgd. Gu6rin et al. (1983) described two recombinants which supported the order as Hal-Phi-Pgd. The recombinants were both HAL-offspring of matings between Hal Nln and Hal Wn animals. The failure of these animals to react to halothane could, however, have resulted from the incomplete penetrance of the Haln gene.
The S locus controls the expression of the A and 0 antigens of the A-O blood group system in pigs by an epistatic interaction (Rasmusen, 1964 andHojny &Hdla, 1965). Two alleles are known, S being dominant over s.
The relationship between A-O blood group phenotypes determined by genes at the S locus and HAL+ animals (Rasmusen & Christian, 1976) was in agreement with the associations found between A-O and H blood group systems (Rasmusen, 1972). Hojny (1974) suggested that this association resulted, indirectly, from the genetic linkage between the H system and the S locus. Rasmusen (1981) proposed the order Phi-Hal-S-H-Pgd on the basis of recombinants between S and H as well as between S and Phi-Hal. Later, two .other reports provided evidence that the S locus is not within the Phi-Pgd region, but adjacent to Phi (Hojny et al., 1985;Van Zeveren et al., 1985). -Recently it has been found that the serum postalbumin-2 (Po2 locus) also belongs to the S-(Phi-Hal-H)-Pgd linkage group and is probably located between the H and Pgd loci (Juneja et al., 1983;Gahne & Juneja, 1985;Cepica et al., 1986).
The aim of this paper is to reconsider the gene order in the linkage group, especially of the Phi and Po2 loci and to establish the haplotypes (including Hal genotypes) in a population of Swiss Landrace pigs. Estimation of recombination frequencies is given elsewhere (V6geli et al., 1988).

Materials and Methods
Description of the data Data for this study came from Swiss Landrace pigs kept at the experimental station of the Institute of Animal Sciences during the period [1983][1984][1985][1986][1987][1988]. The total number of offspring was 1348. The animals came from 165 litters produced by 29 boars and 64 sows over 3 successive generations.

Halothane test
At an age of 8 to 12 weeks the animals were tested for halothane sensitivity by the method of Eikelenboom & Minkema (1974). The anesthetic was a mixture of oxygen and 4% halothane (1.5 liters/min). Negatively reacting animals were exposed for 5 min. In HAL + pigs the anesthesia was withdrawn as soon as the symptoms of hyperthermia (muscular rigidigy, increased heart rate and elevated body temperature) became apparent.

Serological tests
The A and 0 reagents were prepared from normal serum of 2 goats and were used in the hemolytic test. The alloimmune anti-Aw was applied in the dextran agglutination test.
The blood group factors Ha and Hc were tested using two reagents each. One of each exhibited dosage effects, i.e., they hemolysed red blood cells of homozygous (H a / h O , H C / H C ) pigs sooner than those derived from heterozygous (H a / H -, H C / H -) pigs.
The validity of the reaction pattern of these reagents was verified in International Pig Comparison Tests (1984 and 1987, the latter being organized by our laboratory).

Parentage control
Tests for other blood marker systems (B, G, ADA, PGM, P11, P01A, P12 ) were conducted on all animals for the exclusion of incorrect pedigrees.

Haplotyping
The method used in the present study to determine the haplotypes was based on deducing linkage phases involving Hal and marker loci of both the parents and their offspring. A detailed description of the procedure is given by V6geli et al. (1988). Several instances of crossing over were observed in progeny from multiheterozygous parents mated to multihomozygous parents. These were used to determine the order of the loci.

Results
Table I provides a summary of recombinations involving the Hal and Phi loci recovered in progeny from matings in which one parent was at least triply heterozygous and the other doubly or multiply homozygous. All the recombinations are informative with respect to the location of the Phi locus. The structure of parental haplotypes was inferred from various informative matings.
Assuming that the gene order is Phi-Hal-H as suggested by Rasmusen (1981) and not Hal-Phi-H, the first 5 recombinants of the first 3 matings given in Table I would have required the occurrence of a double crossover, i.e., a crossover between Phi and Hal as well as a crossover between Hal and H which is statistically extremely unlikely.
Mating of boar 8888 with female 8849 produced a recombinant (offspring 9925) resulting in an unexpected halothane negative reaction of this offspring. This recombinant could be explained as being a result of double crossover. However, incomplete penetrance of HaM / HaM seems more likely. Unfortunately, the recombinant offspring 9925 was not saved for breeding to determine his actual genotype. In the offspring of animals with Hafn Hain genotype mated to Ha! / HaM, about 10% are classified as HAL- (Gahne & Juneja, 1985.). The failure of one offspring from a total of six to react to halothane could well be the result of incomplete penetrance of the Hal gene. From these considerations the gene order of Hal-Phi-H is suggested. Table II shows most informative recombinants between S, Hal, Phi and H on one side and Po2 and Pgd on the other. All five recombinants are informative in determining the position of the Po2 locus. From these data the gene order is H-Po2-Pgd as proposed by Juneja et al. (1983). If the gene order were Po2-H-Pgd, all 5 recombinants could only have resulted from double crossovers (Phi Î-P 0 2 Î-H-Pgd), which is highly improbable. Table III shows the parents and offspring of 2 litters which include recombinants involving a crossover between loci for Phi and H types. These marker loci are also consistent with a gene order of Phi-H-Po2 as opposed to H-Phi-Po2.

Discussion
The expected Hal genotype of offspring receiving (a) recombinant haplotype (s) can be determined if the sequence between Hal and marker loci has been established. The most likely order of the marker loci including Hal was indicated as S-(Phi-Hal) -(H-Po2)-Pgd by Hojny et al. (1985) and van Zeveren et al. (1985). As these authors did not detect crossing over between Phi and Hal, they could neither prove nor disprove the reverse sequence for the Phi and Hal loci proposed by Gu6rin et al. (1983). However, they confirmed that the two loci are located very close to each other.
The most important contribution of this paper is the evidence that the Phi locus is located, most probably, between Hal and H as proposed by Guérin et al. (1983) and van Zeveren et al. (1988) and not between S and Hal as previously reported by Andresen (1981) and Rasmusen (1981). This location is more firmly established by complex S-Hal-Phi-H-Po2-Pgd haplotypes of the majority of parents and offspring, including recombinants. Probably because of incomplete penetrance of the Hal gene one animal with presumed genotype Hah / HaM failed to react to halothane. Two recombinants (Table 1, offspring 275 and 695) being informative with respect to the location of the Phi locus were classified as HAL + . These two reactors certainly are Hal !l Hal ! homozygotes because the probability of a Hah l HaI N or HalN / Hain pig being falsely tested as HAL + is very low (V6geli et al., 1988).
The data in Tables II and III Tables I, II and III and those contained in earlier publications indicate a gene order S-Hal-Phi-H-Po2-Pgd. The knowledge of the halothane locus and its linkage relationships is already being used in practical animal breeding to reduce the frequency of the Hal ! gene (Gahne & Juneja, 1985;V6geli et al., 1988). Looking to the future, molecular analysis of the halothane linkage group may provide a means for identifying more reliable markers for the stress genes as well as the identity of the halothane gene itself. One step in this development is the assignment of the Hal linkage group to chromosome 6 by in situ hybridization (Davies ef al., 1988).