key: cord-010500-ajmj2hyj authors: ELLEGREN, H.; MIKK, S.; WALLIN, K.; ANDERSSON, L. title: Limited polymorphism at major histocompatibility complex (MHC) loci in the Swedish moose A. alces date: 2008-06-28 journal: Mol Ecol DOI: 10.1111/j.1365-294x.1996.tb00286.x sha: doc_id: 10500 cord_uid: ajmj2hyj The Swedish moose was analysed for genetic variability at major histocompatibility complex (MHC) class I and class II DQA, DQB and DRB loci using restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) techniques. Both methods revealed limited amounts of polymorphism. Since the SSCP analysis concerned an expressed DRB gene it can be concluded that the level of functional MHC class II polymorphism, at least at the DRB locus, is low in Swedish moose. DNA fingerprinting was used to determine if the unusual pattern of low MHC variability could be explained by a low degree of genome‐wide genetic diversity. Hybridizations with two minisatellite probes gave similarity indices somewhat higher than the average for other natural population, but the data suggest that the low MHC variability cannot be explained by a recent population bottleneck. However, since minisatellite sequences evolve more rapidly than MHC sequences, the low levels of MHC diversity may be attributed to a bottleneck of more ancient origin. The selection pressure for MHC variability in moose may also be reduced and we discuss the possibility that its solitary life style may reduce lateral transmission of pathogens in the population. The gene products of the major histocompatibility complex (MHC) system are recognized as key molecules in the immunological recognition of self and nonself (Klein 1986 ). There are two distinct classes of MHC molecules, class I and class II, which are encoded by separate but tightly linked loci. Both are cellsurface glycoproteins which function as presenters of intracellulary processed peptides. In a majority of the species so far studied, MHC represents the most polymorphic gene system found in the genome. Unlike most other polymorphic genes, MHC variability is characterized by the presence of a large number of alleles that occur at intermediate frequencies (Klein 1986 ). This is a pattern expected for loci under balancing selection (Hedrick & Thomson 1983) and it is generally believed that the high degree of genetic variability at MHC Correspondence: Dr Hans Ellegren. Fax: + 4 6 18 504461. E-mail: Hans.Ellegren@bmc.uu.se is maintained by overdominant or frequency-dependent selection (Klein et al. 1993) . Many investigators argue that this relates to the immunological defence against pathogen-induced diseases (Bodmer 1972; Doherty & Zinkemagel1975) , although other selective forces such as mating preference and maternal-fetal interactions have been suggested as well (Hedrick & Thomson 1988; Potts & Wakeland 1993) . It has been suggested that populations which have experienced extensive bottlenecks, leading-to Educed MIX variability, would be particularly vulnerable to infectious disease (O'Brien & Evermann 1988) . Preserving MHC polymorphism has in fact been put forward as being a prime objective in all conservation programs (Hughes 1991) . In line with this, it has been proposed that the extremely low level of MHC variability in the African Cheetah (O'Brien et al. 1985; Yuhki & O'Brien 1990 ) may be related to a marked susceptibility to a feline coronavirus infection in a group of captive individuals (O'Brien et al. 1985) . Severe outbreaks of infectious disease in some other species speculated to possess reduced MHC variability may represent similar situations as well (OBrien & Evermann 1988) . We are involved in a study of the possible association between genetic variation at MHC loci and susceptibility to a lethal disease syndrome, still of unknown origin, among Swedish moose A. alces (Stken et nl. 1993) . We have now used various molecular techniques to assess the amount and characteristics of MHC class I and class I1 variability in Swedish moose and in parallel analysed their genome-wide variability by means of DNA fingerprinting. We show here that these animals exhibit low levels of MHC polymorphism concurrent with a fairly high degree of genomic diversity. Two sets of animals were analysed. First, liver samples were obtained from 10 free-living moose collected in four different areas spread from southern to northern Sweden. Second, liver samples were also derived from 18 free-living individuals shot during the autumn hunt in a 35 kmz area in central Sweden. Apparent relatives were avoided in the latter case by only collecting a single specimen from each shooting location. Pieces of liver were transferred to -20 "C not later than six hours after shooting. We have previously made extensive genetic studies of the MHC system of cattle (e.g. Sigurdardottir et al. 1988). We will make reference to cattle several times in this study since this species represents the closest relative to the moose for which MHC variation previously has been well characterized. All cattle data cited have been obtained from the Swedish Red and White breed. Gnomic DNA was prepared from blood and liver as previously described (Ellegren et al. 1991) . Southern blots of TiqI, PVuII, EcoRI or HindIII digests (10 pg) were prepared foilowing standard procedures, i.e. electrophoretic separation of fragments in 1.0% agarose gels and subsequent transfer to nylon filters (Biodyne B) at aibaline conditions. Hybridization was carried out according to Mariani et al. (1992) with a final washing stringency of 0.7 x SSC; 0.1% SDS at + 58 "C. We used four human MHC cDNA clones as hybridization probes: (Gustafsson et al. 1984) . The three latter probes correspond to h4HC class II genes. Filters were prepared as above except that the DNA was digested with Hiiifl and that 0.7% agarose gels were used. We employed two commonly used minisatelhe probes, MS6 (Jeffreys et al. 1985 ) and a 952 bp BstNI fragment from phage M13 (Vassart et nl. 1987) . Hybridization was performed in the absence of competitor DNA and filters were washed at a final stringency of 2 x SSC; 0.1% SDS at + 55 "C. Exon two of the MHC DRB gene was amplified by PCR using the cattle specific primers LA31 and LA32 (Sigurdardottir et al. 1991) . The 20-pL reactions contained 0.2 mM dNTP, 0.5 p~ of each primer, 25 ng of genomic DNA and 1 U Taq polymerase. A total of 30 cycles was performed, the three first had + 95 "C for 30 s, + 50 "C for 10 s and + 72 "C for 45 s, the following 27 had + 94 "C for 10 s, + 50 "C for 10 s and + 72 "C for 30 s. The PCR was performed on a Perkin-Elmer-Cetus 9600 instrument. The SSCP analysis essentially followed Maekawa et al. (1992) . Briefly, PCR products were mixed with a 1.5 volume denaturing solution containing 95% formamide, 20-m~ EDTA, 0.05% BFB and 0.05% xylene cyanol, and were incubated at + 80 "C for 10 min. Samples were subsequently applied to PhastSystem gradient gels 8-25 (previously prerunned at 250 V, 10 mA and 2.5 W for 60 Vh) and electrophoresed with native buffer strips at + 15 "C in a PhastSystem device (Pharmacia, Uppsala) for 330 Vh (using the same settings as above). After separation, the gels were silver-stained with the PhastSystem silver staining kit. Human cDNA clones encoding HLA class I (locus not determined), HLA-DQA, HLA-DQB and HL4-DRB genes were hybridized to moose TqI, PvuII, EcoRI and HindIII digests. The screening was performed on 10 unrelated individuals sampled at various sites in Sweden. The outcome of the experiments is summarized in Table 1 . Clearly, the levels of polymorphism were very limited. Completely monomorphic hybridization patterns were revealed in seven out of 16 probe/enzyme combinations. Furthermore, the seven polymorphic class Il probe/enzyme combinations did most likely only detect two different D Q haplotypes. This conclusion was reached on the basis of that the polymorphic fragments, only one or two in each case, consistently were present in the same @ 1996 Blackwell Science Ltd, Molecular Ecology, 5,3-9 Table 1 Summary of the moose MHC RFLP analysis. The number of polymorphic fragments are indicated for each probe/ enzyme combination with the total number of fragments in parentheses. Obviously cross-hybridizing fragments were excluded from the total numbers Enzyme Locus raqr Paun Class I 1(II) four individuals in all seven combinations. The variability seen with the class I probe was similarly low, i.e. monomorphic pattern with two enzymes and one polymorphc fragment with two other enzymes. The latter polymorphisms represented different haplotypes as compared to the class I1 polymorphisms (the polymorphic TuqI fragment was present in five individuals whereas the polymorphic EcoRI fragment only occurred in two individuals). In an extended screening we analysed 18 additiocal moose sampled in central Sweden. The screening was restricted to DQB and DRB probes hybridized to PuuII digests. As for the first 10 individuals, a completely monomorphic pattern was revealed with the DRB probe. Four out of five hybridization fragments specifically detected by the DQB probe were polymorphic in this material but could be interpreted as a simple two-allele polymorphism (Fig. la) . One allele was composed of 2.4-highly significant difference (xZ1 = 46.2, P < 0.001). Furthermore, in a study of bovine MHC class I polymorphism, Lindberg Q distinguished no less than 21 PvuII haplotypes in a sample of 5O unrelated individuals. There were no more than two class I haplotypes seen with any enzyme in moose. As an illustration of the varying levels of MHC polymorphism in cattle and moose, a blot with bovine PVuII digests hybridized with the same human DQB probe as employed in the present study is shown in Fig. I@) . Clearly, the degree of polymorphism differs considerably between the two species. The primers LA31 and LA32 specifically amplify exon two of the bovine DRB3 gene, the most predominantly expressed and the most polymorphic DRB gene in cattle. The primer pair was used in amplifications with moose DNA and a single distinct PCR product was revealed for each individual. The size of the amplified product exactly corresponded to that obtained for the bovine DRB3 gene (297 bp) and subsequent sequence analysis made evident that the product represented a moose MHC DRB gene, denoted DRBl (Mikko C Andersson 1995) . It can be concluded that this locus constitutes an MHC gene of func- tional importance since it is the pmiominantly expressed DRB locus in moose according to RT-PCR analysis (Mikko & Anderson 1995) . SSCP analysis of the DRBl gene amplified from 16 moose identified three different alleles (Fig. 2a) . One allele was obviously rare since it was only found in one heterozygous animal. The frequencies of the other two alleles were 053 and 0.41, respectively (H-= 0.55). Again this indicates that the moose has limited MHC class Il poly-morphism. A sample of cattle analysed with the same SSCP methodology (i.e. using the same primers) is shown in Fig. 2b . A characteristic feature of the MHC is the presence of strong linkage disequilibrium between loci. This was also evident from the present data. Using the method of Hill (1974), a significant linkage disequilibrium between the moose DQB RFLP and the DRB SSCP alleles was observed (D = 0.17; P c 0.05). The relative amount of disequilibrium (Hedrick et al. 1978 ) was estimated at 0.68. We carried out DNA fingerprinting using two minisatellite probes and Hinff digested DNA to analyse the genomewide variability of Swedish moose. Statistics derived from these experiments are given in Table 2 and a representative autoradiograph is shown in Fig. 3 . Similarity indices (D) calculated from hybridizations with MS6 and MI3 were 0.46 and 0.49, respectively. These values are somewhat higher than that previously =ported for a number of out- and is more similar to that obtained for several domestic animals (e.g. Georges et al. 1988 ). We have investigated the genetic variability of moose MHC class I and class II genes by means of (i) RFLP analy- technique that is one of the most sensitive methods for detecting point mutations. SsCp analysis relies on the fact that the mobility rate of a single-stranded DNA molecule in a nondenaturing gel is not only determined by its size but also by its nudeotide sequence, which, in turn, govems its threedm * ensional structure (Orita ef al. 1989) . Even a single nudeotide substitution may slightly alter this structure leading to a differential migration pattern. It has been estimated that SSCP can distinguish between 7096 and 95% of all possible mutations in PCR products 0 1996 Blackwell Science Ltd, Molecular Ecology, 5,3-9 < 200 bp and about 50% for those < 400 bp (Michaud et al. 1992; Sheffield et al. 1992 ). The finding of low levels of MHC variability in Swedish moose is thus supported by the inherent sensitivity of the SSCP technique. The fact that an expressed moose MHC gene was analysed rules out the possibility that the limited variability could be explained by a reduced selection pressure at a locus of no functional significance. The sequence analysed by SSCP, exon two of DRB, encodes the antigen recognition site in the first extracellular domain of dass II / 3 chain genes. In other species, the genetic polymorphism of class n gene products is concentrated to particular amino acids involved in antigen binding encoded by this exon (Brown et al. 1988) . A characteristic feature of this polymorphism, as known from various species, is that there is a considerable amino acid divergence between alleles (Klein 1986; Andersson & Davies 1994) . In an accompanying study we have shown that the Swedish as well as the Canadian moose does not only possess limited amounts of DRB poly&orphism but also has minor sequence variations between alleles (Mikko & Andersson 1995) . What is the causal basis for the low levels of MHC polymorphism among the moose analysed in this study? A possible explanation would be that a previous bottleneck could have reduced the genetic variability. Similar to the situation for the Scandinavian beaver population (EIIegren et nl. 19!33), the size of the Swedish moose population was reduced due to over-hunting in the last century. The pop ulation has subsequently recovered and has increased dramatically during the 20th century, currently harbouring some 400 OOO individuals. However, the extent of the bottleneck in the 19th century is uncertain (cf. Ryman et al. 1980) and our data strongly argue against a severe reduction since DNA fingerprinting patterns were far from monomorphic. The observed band-sharing probabilities (0.4.49) are somewhat higher than that previously found among outbred natural populations (e.g. Reeve et al. 1990 ), but do not indicate a dramatic loss of genetic variability. Similarly, Ryman et al. (1980) analysed allozyme variability in a large number of moose sampled at various localities in Scandinavia and found a mean heterozygosity, within each popdation, of 0.020. Again Cheetah, Menotti-Raymond & O'Brien (1993) calculated that the moderate DNA fingerprinting variability found among modem Cheetahs could have been reconstituted (from complete homozygosity) in the order of 10 OOO years. Although the DNA fingerprinting variability in our moose sample was higher than among the Cheetahs, calculations similar to those made by Menotti-Raymond and OBrien are consistent with that the Swedish moose may have experienced a severe bottleneck in the last 10 OOCL50 OOO years. If so, extensive MHC variability is not to be expected among contemporary moose since the mutation rate at MHC loci is significantly lower than that at minisatellite loci. As evident from interspecific comparisons, extensive MHC polymorphism reflects the accumulation of mutations during millions of years of evolution (Klein et al. 1993) . Besides the possible effects caused by an ancient bottleneck, the low levels of moose MHC variability could also be due to a relatively weak selection pressure for p l ymorphism at these loci. There is a clear difference between the moose and cattle as regards their degrees of MHC polymorphism in relation to their genome-wide genetic variability measured by DNA fingerprinting. The DNA fingerprinting variability of moose is similar to that among several domestic animals, including cattle (Georges et nl. 1988), and is significantly lower than that in humans. In contrast, the degree of MHC polymorphism in moose is much lower than that in both cattle and humans. Differences in the intensity of pathogen-driven, balancing selection affecting moose and cattle may contribute to the marked differences in their degree of MHC diversity. Domestic cattle are kept in dense herds which obviously facilitates the transmission of pathogens between animals. The moose, on the other hand, has a rather solitary life style. Males' close contacts with other animals are probably restricted to the reproduction period, a situation that would reduce the lateral transmission of pathogens in the population. The Swedish moose consequently provides an additional example of a viable population with restricted MHC polymorphism. Slade (1992) noticed that three marine mammals from two different orders (the elephant seal, the sei whale and the fin whale) exhibit low degrees of MHC variability despite no apparent loss of allozyme heterozygosity. He proposed that this could be due to a reduced balancing selection pressure in the marine environment, a possible consequence of decreased exposure to pathogens. Trowsdale et nl. (1989) , who analysed the two whale species, argued that the aquatic environment would impose less contact between the animals compared to a terrestrial environment and that this would hamper the spread of pathogens in the population. A similar but controversial idea has been invoked to explain the apparent lack of MHC class I polymorphism in Syrian hamsters (Darden & Streilein 1984) ; the solitary life style of this species could act as a barrier for pathogen transmission as well (McGuire et nl. 1985) . In this context it could be noted that beavers, of which at least three European populations appear to be devoid of MHC polymorphism (Ellegren et nl. 1993), also have a rather isolated life style. Although they live in family groups and dispersal may occur in natal drainage systems, large land areas separating different river systems can be seen as barriers for long-range dispersal and hence also for lateral transfer of pathogens. Most authors, including us, have interpreted the presence of reduced levels of MHC diversity in certain species to be due to previous bottlenecks or relaxed pathogen-driven selection. However, it should be noted that the hypothesis of pathogen-driven selection for MHC polymorphism has not yet been confirmed by experimental data, nor has it been shown that the level of MHC diversity influence the long term survival of natural populations. It is dear that data from a broad range of species will be required to shed light on this important question. 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Evolution Both a and fi chains of HLA-DC class I1 histocompatibility antigens display extensive polymorphism in their amineterminal domains Analysis ofthe Eficiency of Single Base Substitution Detection by SSCP Restriction fragment length polymorphism of DQ and DR class I1 genes of the bovine major histocompatibility complex Cloning and sequence analysis of 14 DRB alleles of the bovine MHC by using the polymerase chain reaction Limited MHC polymorphism in the southern elephant seal: implications for MHC evolution and marine mammal population biology An erosivelulcerative alimentary disease of undetermined etiology in Swedish moose Limited MHC polymorphism in whales A sequence in M13 phage detects hypervariable minisatellites in human and animal DNA Large mammals are genetically less variable DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history This work is part of long-term project aimed at understanding the evolution and polymorphism of MHC in different mammals. Hans Ellegren is associate professor, Sofia Mikko is PhD student and Leif Andersson is professor in molecular and disease genetics at the Swedish University of Agricultural Sciences in Uppsala. This particular study arose from a collaboration with Dr Kjell Wallin at the same university who has a keen interest in the biology of moose.