- Email: [email protected]
Evolutionary implications of haemoglobin polymorphism in domesticated and wild sheep
SmallRuminant Research, 4 ( 1991 ) 315-322
315
Elsevier Science Publishers B.V., Amsterdam
Evolutionary implications of haemoglobin polymorphism in domesticated and wild sheep S. W a n g a'b, W . C . F o o t e b a n d T . D . B u n c h b
aCommission for Integrated Survey of Natural Resources, The Chinese Academy of Sciences, Beijing, People's Rep. of China bDepar~ment of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-4815, USA (Accepted 20 May 1990)
ABSTRACT Wang, S., Foote, W.C. and Bunch, T.D., 1991. Evolutionary implications of haemoglobin polymorphism in domesticated and wild sheep. Small Rumin. Res., 4:315-322. The distribution of haemoglobin polymorphism was investigated in 11 breeds of domesticated and three genetic groups of wild sheep in the USA. Hbfl B was more frequent than HbflA in most of the domesticated breeds with a pooled frequency of 0.71. The HbflB gene frequencies were: 0.95 (Barbados), 0.45 (Booroola), 0.77 (Columbia), 0.40 (Finnsheep), 0.87 (Hampshire), 0.88 (Karakul), 0.98 (Rambcuillet flock 1 ), 0.93 (Rambouillet flock 2), 0.12 (Romanov), 0.50 (St. Croix flock 1 ), 0.63 (St. Croix flock 2), 1.00 (Suffolk flock 1 ), 0.86 (Suffolk flock 2) and 0.86 (Targhee). All the wild sheep tested [Argali× European Mouflon cross (Ovis ammon × Ovis m usimon), Asiatic Mouflon × European Mouflon cross (Ovis orientalis × Ovis musimon ) and Desert Bighorn (Ovis canadensis) ] were monomorphic for HbflB. The monomorphism of HbflB of wild sheep and the higher frequency of Hbfla than HbflA in domesticated sheep indicates that HbflA probably occurred after sheep were domesticated. The wide variation in haemoglobin polymorphism within and between breeds may have resulted from a combination of genetic drift and selection.
INTRODUCTION
Since first reported in 1955 (Blunt and Huisman, 1975 ), haemoglobin (Hb) polymorphism has been found in most breeds of sheep and its distribution investigated extensively (Agar et al., 1972; Blunt and Huisman, 1975; Bunch and Foote, 1976; Reddy et al., 1988; Rizzi et al., 1988). It was reported that haemoglobin variants differ in oxygen affinity and therefore may differ in adaptive values with respect to reproductive performance and intestinal parasite susceptability (Agar et al., 1972; Blunt and Huisman, 1975 ). It was also noticed that the distribution of haemoglobin variants is related to geographic environments (Agar et al., 1972 ). Some evidence indicates that haemoglobin types may have influence on certain physiological aspects, such as the concen0921-4488/91/$03.50
© 1 9 9 1 - Elsevier Science Publishers B.V.
316
s. WANG ET AL.
tration of reduced glutathione in erythrocytes and whole blood, which has been correlated with daily milk yield (Rizzi et al., 1988 ). Haemoglobin as a genetic marker, combined with other blood proteins, has also been used to study the relationship between breeds of sheep (Manwell and Baker, 1977; Buis and Tucker, 1983; Iovenko, 1987 ), and is valuable in pedigree control in breeding programmes (Baker and Manwell, 1983 ). This paper presents the distribution of Hb polymorphism among populations of domesticated and wild sheep in the USA and discusses the implications of these findings on sheep evolution. MATERIALS AND METHODS
Domesticated sheep (447) from 11 breeds (Barbados, Booroola, Columbia, Finnsheep, Hampshire, Karakul, Rambouillet, Romanov, St. Croix, Suffolk and Targhee) and 92 wild sheep from three genetic groups [Argali× European Mouflon cross (Ovis ammon X Ovis musimon), Asiatic Mouflon × European Mouflon cross (Ovis orientalis × Ovis musimon) and Desert Bighorn (Ovis canadensis nelsoni)] were sampled. These sheep populations were sampled from various areas in the USA (Table 1 ). Vertical polyacrylamide gel electrophoresis was used to separate the haemoglobin types. Procedures were according to Andrew (1986) with certain revisions. Stacking gel was made of 0.06 M Tris-HC1 buffer (pH 6.8 ) and a gel concentration of 3% (C = 20). The separating gel was composed of 0.37 M Tris-HC1 buffer (pH 8.9) and a gel concentration of 7% ( C = 2 . 5 ) . The electrophoresis solution was 0.05 M Tris-glycine buffer (pH 8.9). Electrophoresis was run under a constant voltage of 300 V for approximately 3 h. The gel was stained with 0.4% Coomassie Blue R250 in a solution of 9 vols ethanol, 9 vols water and 2 vols acetic acid. Jugular vein blood was collected and heparin or potassium oxalate was used as the anti-coagulant. Red blood cells were washed three times with a 0.9% NaC1 solution. Erythrocyte lysates were obtained by adding 2 vols of distilled water and centrifuged at 2700 rpm for 20 minutes at room temperature. The supernatant was removed and stored at - 20 ° C. Lysates were diluted 20 times and glycerol ( 10% in final concentration) or sucrose (20% in final concentration) was added. Three/tl of this preparation were applied to each sample well in the gel slab. Standard samples of sheep haemoglobin types were provided by Dr. C.M.T. Penedo, Serology Laboratory, School of Veterinary Science, University of California at Davis. These references were also used for the identification of haemoglobin types of wild sheep. Frequencies of haemoglobin alleles were calculated according to Nix et al. (1969) and Lauvergne ( 1989):
EVOLUTIONARY IMPLICATIONSOF HAEMOGLOBIN POLYMORPHISM IN SHEEP
317
f = 2AA-t-.AB/ 2 (No. of animals sampled ) where AA and AB are the numbers of haemoglobin phenotypes HbflAHbflA and HbflAHbflB, respectively. The Chi-square method was used to test significant differences in allelic frequencies between sheep populations and the deviation from Hardy-Weinberg equilibrium. RESULTS
Phenotypic and allelic frequencies for the populations of wild and domesticated sheep sampled are in Table 1. Both Hbfl A and Hbfl B alleles were observed in nearly all domesticated sheep populations. The HbflB gene was more frequent than the HbflA gene in most cases except in Romanov, Finnsheep TABLE 1 Frequency of alleles at the haemoglobin (Hbfl) locus in domesticated and wild sheep in USA 1 Populations
Symbols
Phenotypes
Gene frequencies
N
AA
AB
BB
Hbflg
Hbfl a
Texas Nebraska Dubois, Idaho Nebraska Utah Texas Utah Dubois, Idaho Nebraska Northern Utah USVirgin Islands Southern Idaho Northern Utah Dubois, Idaho
20 40 35 38 41 20 30 34 30 35 30 30 31 33
0 13 1 14 1 0 0 0 23 8 7 0 0 1
2 18 14 18 9 5 1 5 7 19 8 0 9 7
18 9 20 6 31 15 29 29 0 8 15 30 22 25
0.05 0.55 0.23 0.60 0.13 0.12 0.02 0.07 0.88 0.50 0.37 0.00 0.15 0.14 0.29
0.95 0.45 0.77 0.40 0.87 0.88 0.98 0.93 0.12 0.50 0.63 1.00 0.85 0.86 0.71
AGMO
Utah
33
0
0
33
0.00
1.00
ARMM
Texas
28
0
0
28
0.00
1.00
DBIG
Utah and Arizona
31
0
0
31
0.00
1.00
Domesticated Sheep Barbados BARB Booroola BOOR Columbia COLM Finnsheel9 FINN Hampshi:re HAMP Karakul KARA Rambouillet- 1 RAMB 1 Rambouillet-2 RAMB2 Romano~ ROMA St. Croix- 1 STCX 1 St. Croix-2 STCX2 Suffolk- 1 SUFK1 Suffolk-2 SUFK2 Targhee TARG Pooled average frequencies. Wild Sheep Argali × European Mouflon Asiatic Mouflon × European Mouflon Desert Bighorn
Location
ISignificance of differences explained in text.
318
S. W A N G ET AL
<
o.
o ~D <
c~c~
{D < [..
a~ od a~
0
o
< <
©
< II
~j
o
A
z v
~,
f',l ..1 <
o "~
..~ 0 (D
"
0
~
g
v~
8~
EVOLUTIONARY IMPLICATIONS OF HAEMOGLOBIN POLYMORPHISM IN SHEEP
319
and Booroola. Average frequency of the Hbfl a allele for domesticated sheep was 0.71. The Hbfl B gene frequencies among populations of domesticated sheep fluctuated between 0.12 and 1.00. All wild sheep sampled in this survey were monomorphic for Hbfl B. One significant ( P < 0.05 ) deviation from the Hardy-Weinberg equilibrium was observed in St. Croix flock 2. This deviation was due to the lack of heterozygotes. There were no predictable relationships between breeds in the allelic frequencies of haemoglobin. Distantly related breeds may share the same types of alleles with similar frequencies, such as the Karakul compared to Targhee and Suffolk, and the Booroola to St. Croix (Table 1 ), while breeds sharing close relationship in origin and development may differ significantly ( P < 0.05 ), such as the Columbia compared to Rambouillet (Tables 1 and 2 ). Significant differences (Table 2) existed in the Hb gene distribution between Barbados vs. Finnsheep, Romanov vs. all other sheep populations ( P < 0.01 ), St. Croix vs. Rambouillet and Suffolk ( P < 0.01 ), and Targhee vs. Booroola ( P < 0 . 0 1 ) . Significant differences ( P < 0.05 ) were also observed between populations (flocks) of the Suffolk breed but not between those of St. Croix and Rambouillet breeds (Table 2 ). DISCUSSION
Nadler et al. ( 1971 ) reported that the wild sheep from Asia (Iran), Europe and North America (Ovis dalli, Ovis musimon and Ovis canadensis) were all Hbfl B type. Lay et al. ( 1971 ) and Bunch and Foote (1976) observed Hbfl a monomorphism in wild Iranian sheep (Ovis orientalis, Ovis vignei and the hybrids of the two). The Hbfl B was the only allele in wild sheep (Ovis ammon× Ovis musimon, Ovis orientalis× Ovis musimon and Ovis canadensis) observed in this study and confirms what has been previously reported. Agar et al. (1972) reviewed the Hb polymorphic distribution of sheep in many locations over the world, and reported that more than two thirds of the populations surveyed were more frequent in Hbfl B than Hbfl A. This was also observed in indigenous domestic breeds (Tan sheep and Mongolian sheep) in China (Wang, 1983, personal communication), and the major domestic sheep breeds in Iran (Bunch and Foote, 1976), the Nellore sheep in India (Reddy et al., 1988 ), the Askania and Tsigai sheep in USSR (Iovenko, 1987 ) and other native sheep breeds in Europe (Rizzi et al., 1988 ). Lay et al. ( 1971 ), Bunch et al. (1978) and Valdez et al. (1978) hypothesized that wild sheep gave rise to the Hbfl A allele, which most likely evolved after the domestication of sheep. Evidence for this conclusion was based upon the fact that the Hbfl A allele was not found in wild sheep and Hbfl B was more frequent than Hbfl g in most domesticated sheep. The molecular structure of haemoglobin seems to support this hypothesis.
320
s. WANG ETAL.
The Hbfl a of wild sheep is electrophoretically similar to Hbfl B of domesticated sheep. However, wild sheep Hbfl B differs from domesticated sheep Hbfl B (beta-chain) at five amino acids and from domesticated sheep Hbfl A only at two amino acids. These different amino acid positions do not overlap with each other (Manwell and Baker, 1976). According to the constancy of the amino acid substitution rate (Kimura, 1983 ), Hbfl A must have occurred later than Hbfl B, since it involved less amino acid substitutions. It is likely that the Hbfl A allele is now in the process of spreading throughout all breeds of domesticated sheep. The extensive variation of the haemoglobin polymorphic distribution in domesticated animals may be attributed to natural selection under different environments, differential selection and management and genetic drift. From our data we could not determine their relative influence. However, the rand o m pattern of allelic distribution might indicate that genetic drift is an important factor in causing variation. As shown by the results, relationships between sheep breeds in haemoglobin distribution in terms of alleles and their frequencies are not predictable (Table 1 ). Furthermore, flocks within the same breed may or may not show significant differences in allelic frequencies, and this seems not to be related to the variation in environments (Tables 1 and 2). Significant differences occurred between flocks of the same breed located in the same geographical region (two flocks of Suffolk, one from Northern Utah and the other from Southern Idaho). However, there was no significant difference between the two flocks of St. Croix and between the two flocks of Rambouillet. These two flocks in each breed were located in areas where the environments were quite different. One flock from the St. Croix breed (STCX 1 ) was located in Northern Utah. The flock was established in 1975 and propagated from two rams and 22 ewes from the Island of St. Croix in the Caribbean. The other St. Croix flock (STCX2) was located on St. Croix Islands and some of the animals are from the source flocks for STCX 1. Haemoglobin polymorphic distribution among flocks did not differ from each other (Table 1 ). CONCLUSION
The observation that allelic frequency ofHbfl B was greater than for the HbflA allele in most domesticated sheep breeds and was the only allele that existed in genetic groups of wild sheep, supports the hypothesis that the Hbfl A allele arose after domestication of sheep. The wide variation in allelic distribution among domesticated sheep populations most likely resulted from selection and genetic drift. The random pattern of haemoglobin polymorphic distribution indicates that genetic drift is an important factor leading to the polymorphic frequency differentiation.
EVOLUTIONARY IMPLICATIONS OF HAEMOGLOBIN POLYMORPH1SM IN SHEEP
321
ACKNOWLEDGMENTS W e are :grateful to Drs. C . M . T . P e n e d o , R. S w e n s o n , D. M a t t h e w s , S. Wildeus, K. E r c a n b r a c k , L. Y o u n g a n d M. S h e l t o n for p r o v i d i n g h a e m o g l o b i n s a m p l e s fi~r this study. Special t h a n k s to Mrs. A. M a c i u l i s for l a b o r a t o r y ass i s t a n c e a n d to M r . R . C . E v a n s for h e l p in collecting s a m p l e s . T h i s p a p e r is p u b l i s h e d as U t a h A g r i c u l t u r a l E x p e r i m e n t S t a t i o n J o u r n a l p a p e r n u m b e r 3840.
REFERENCES Andrew, A.T., 1986. Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications, 2nd Edn., Clarendon Press, Oxford, pp. 79-92. Agar, N.S., Evans, J.V. and Roberts, J., 1972. Red blood cell potassium and haemoglobin polymorphisrn in sheep. Anim. Breeding Abstr. 40 (3): 407-436. Baker, C.M.A. and Manwell, C., 1983. Electrophoretic variation of erythrocyte enzymes of domesticated mammals. In: N.S. Agar and P.G. Board (Editors), Red Blood Cells of Domestic Mammals, Elsevier North-Holland, Amsterdam, pp. 367-412. Blunt, M.H. and Huisman, T.H.J., 1975. The hemoglobin of sheep. In: M.H. Blunt (Editor), The Blood of Sheep, Springer-Verlag, New York, pp. 155-183. Buis, R.C. and Tucker, E.M., 1983. Relationships between rare breeds of sheep in Netherlands as based on blood typing. Anim. Blood Groups Biochem. Gen. 14:17-26. Bunch, T.D. and Foote, W.C., 1976. Chromosomes, hemoglobin, and transferrins of Iranian domesti,z sheep. J. Hered. 67:167-170. Bunch, T.D., Nguyen, T.C. and Lauvergne, J.J., 1978. Hemoglobin of the Corsica-Sardinian Mouflon (Ovis musimon) and their implications for the origin of HbA in domestic sheep (Ovis aries). Ann. Genet. Sel. Anim. 10: 503-506. Iovenko, V N., 1987. Comparison of the genetic structures of sheep populations using biochemical polymorphisms. Biol. 12: 35-39. (Anim. Breed. Abstr. 57: 1684). Kimura, M., 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. Lauvergne, J.J. (ed.), 1989. Standardized genetic nomenclature for sheep and goats 1987: Loci for visible traits other than colour and blood and milk polymorphisms. Proc. COGNOSAG Workshop July 21-26, 1987, Manosque, Bureau des Ressources Genetiques, Paris, pp. 184. Lay, D.M., Nadler, C.F. and Hassinger, J.D., 1971. The transferrins and hemoglobin of wild Iranian sheep. Comp. Biochem. Physiol. 40B: 521-529. Manwell, C. and Baker, C.M.A., 1976. Protein polymorphisms in domesticated species: Evidence for hybrid origin? In: S. Karlin and E. Nevo (Editors), Population Genetics and Ecology, Academic Press, New York, NY, USA, pp. 105-130. Manwell, C. and Baker, C.M.A., 1977. Genetic distance between the Australian Merino and the Poll Dorset sheep. Genet. Res. 29: 239-253. Nadler, C.F., Woolf, A. and Harris, K.E., 1971. The transferrins and hemoglobin of Bighorn sheep ( Ovis canadensis), Dall sheep ( Ovis dalli), and Mouflon ( Ovis musimon). Comp. Biochem. Physiol. 40B: 567-570. Nix, C.E., Bogart, R. and Price, D.A., 1969. Genetics of plasma transferrins in five breeds of Sheep. J. Hered. 60: 97-100. Reddy, V.P., Reddy, V.R.C. and Reddy, K.K., 1988. Genetics of haemoglobin types and their
322
s. WANG ET AL.
influence on growth traits in Nellore and Nellore X Dorset half bred sheep. Ind. J. Anim. Sci. 58: 1234-1236. Rizzi, R., Caroli, A., Bolla, P., Acciaioli, A. and Pagnacco, G., 1988. Variability of reduced glutathione levels in Masseses ewes and its effect on daily milk production. J. Dairy Res. 55: 345-353. Valdez, R., Nadler, C.F. and Bunch, T.D., 1978. Evolution of wild sheep in Iran. Evolution 32: 56-72. RESUMEN Wang, S., Foote, W.C. y Bunch, T.D., 1991. Implicationes evolutionair del polimorfismo en la hemoglobin en razas dom6sticas y salvajes ovejas. Small Rumin. Res., 4:315-322. La distribuci6n del polimorfismo en la hemoglobina fue investigado en 11 razas dom6sticas y tres grupos gen6ticos de ovejas saivajes en los Estados Unidos de Norteam6rica. Hbfl Bfue mas frecuente que HbflA en la mayoria de las razas dom6sticas con frecuencia promedio de 0.71. Las frecuencias del gen HbflB fueron: 0.95 (Barbados), 0.45 (Booroola), 0.77 (Columbia), 0.40 (Finnsheep), 0.87 (Hampshire), 0.88 (Karakul), 0.98 (Rambouillet flock 1), 0.93 (Rambouillet flock 2), 0.12 (Romanov), 0.50 (St. Croix flock 1), 0.63 (St. Croix flock 2), 1.00 (Suffolk flock 1 ), 0.86 (Suffolk flock 2) y 0.86 (Targhee). Todas las ovejas salvajes examinadas [Cruza de Argali X Mouflon Europea (Ovis ammon X Ovis musimon), cruza de Mouflon Asiatica × Mouflon Europea (Ovis orientalis × Ovis musimon) y Desert Bighorn (Ovis canadensis) ] fueron monom6rficas para HbflB. El monomorfismo de HbflBen ovejas salvajes y la alta frecuencia de Hbfl B comparada con HbflA en ovejas dom6sticas indica que HbflA probablemente se present6 despu6s de la domesticaci6n de la oveja. La amplia variaci6n del polimorfismo en la hemoglobina dentro de las razas y entre las razas puede haber sido consecuencia de una combinaci6n de migraci6n gen6tica al azar y selecci6n.
315
Elsevier Science Publishers B.V., Amsterdam
Evolutionary implications of haemoglobin polymorphism in domesticated and wild sheep S. W a n g a'b, W . C . F o o t e b a n d T . D . B u n c h b
aCommission for Integrated Survey of Natural Resources, The Chinese Academy of Sciences, Beijing, People's Rep. of China bDepar~ment of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-4815, USA (Accepted 20 May 1990)
ABSTRACT Wang, S., Foote, W.C. and Bunch, T.D., 1991. Evolutionary implications of haemoglobin polymorphism in domesticated and wild sheep. Small Rumin. Res., 4:315-322. The distribution of haemoglobin polymorphism was investigated in 11 breeds of domesticated and three genetic groups of wild sheep in the USA. Hbfl B was more frequent than HbflA in most of the domesticated breeds with a pooled frequency of 0.71. The HbflB gene frequencies were: 0.95 (Barbados), 0.45 (Booroola), 0.77 (Columbia), 0.40 (Finnsheep), 0.87 (Hampshire), 0.88 (Karakul), 0.98 (Rambcuillet flock 1 ), 0.93 (Rambouillet flock 2), 0.12 (Romanov), 0.50 (St. Croix flock 1 ), 0.63 (St. Croix flock 2), 1.00 (Suffolk flock 1 ), 0.86 (Suffolk flock 2) and 0.86 (Targhee). All the wild sheep tested [Argali× European Mouflon cross (Ovis ammon × Ovis m usimon), Asiatic Mouflon × European Mouflon cross (Ovis orientalis × Ovis musimon ) and Desert Bighorn (Ovis canadensis) ] were monomorphic for HbflB. The monomorphism of HbflB of wild sheep and the higher frequency of Hbfla than HbflA in domesticated sheep indicates that HbflA probably occurred after sheep were domesticated. The wide variation in haemoglobin polymorphism within and between breeds may have resulted from a combination of genetic drift and selection.
INTRODUCTION
Since first reported in 1955 (Blunt and Huisman, 1975 ), haemoglobin (Hb) polymorphism has been found in most breeds of sheep and its distribution investigated extensively (Agar et al., 1972; Blunt and Huisman, 1975; Bunch and Foote, 1976; Reddy et al., 1988; Rizzi et al., 1988). It was reported that haemoglobin variants differ in oxygen affinity and therefore may differ in adaptive values with respect to reproductive performance and intestinal parasite susceptability (Agar et al., 1972; Blunt and Huisman, 1975 ). It was also noticed that the distribution of haemoglobin variants is related to geographic environments (Agar et al., 1972 ). Some evidence indicates that haemoglobin types may have influence on certain physiological aspects, such as the concen0921-4488/91/$03.50
© 1 9 9 1 - Elsevier Science Publishers B.V.
316
s. WANG ET AL.
tration of reduced glutathione in erythrocytes and whole blood, which has been correlated with daily milk yield (Rizzi et al., 1988 ). Haemoglobin as a genetic marker, combined with other blood proteins, has also been used to study the relationship between breeds of sheep (Manwell and Baker, 1977; Buis and Tucker, 1983; Iovenko, 1987 ), and is valuable in pedigree control in breeding programmes (Baker and Manwell, 1983 ). This paper presents the distribution of Hb polymorphism among populations of domesticated and wild sheep in the USA and discusses the implications of these findings on sheep evolution. MATERIALS AND METHODS
Domesticated sheep (447) from 11 breeds (Barbados, Booroola, Columbia, Finnsheep, Hampshire, Karakul, Rambouillet, Romanov, St. Croix, Suffolk and Targhee) and 92 wild sheep from three genetic groups [Argali× European Mouflon cross (Ovis ammon X Ovis musimon), Asiatic Mouflon × European Mouflon cross (Ovis orientalis × Ovis musimon) and Desert Bighorn (Ovis canadensis nelsoni)] were sampled. These sheep populations were sampled from various areas in the USA (Table 1 ). Vertical polyacrylamide gel electrophoresis was used to separate the haemoglobin types. Procedures were according to Andrew (1986) with certain revisions. Stacking gel was made of 0.06 M Tris-HC1 buffer (pH 6.8 ) and a gel concentration of 3% (C = 20). The separating gel was composed of 0.37 M Tris-HC1 buffer (pH 8.9) and a gel concentration of 7% ( C = 2 . 5 ) . The electrophoresis solution was 0.05 M Tris-glycine buffer (pH 8.9). Electrophoresis was run under a constant voltage of 300 V for approximately 3 h. The gel was stained with 0.4% Coomassie Blue R250 in a solution of 9 vols ethanol, 9 vols water and 2 vols acetic acid. Jugular vein blood was collected and heparin or potassium oxalate was used as the anti-coagulant. Red blood cells were washed three times with a 0.9% NaC1 solution. Erythrocyte lysates were obtained by adding 2 vols of distilled water and centrifuged at 2700 rpm for 20 minutes at room temperature. The supernatant was removed and stored at - 20 ° C. Lysates were diluted 20 times and glycerol ( 10% in final concentration) or sucrose (20% in final concentration) was added. Three/tl of this preparation were applied to each sample well in the gel slab. Standard samples of sheep haemoglobin types were provided by Dr. C.M.T. Penedo, Serology Laboratory, School of Veterinary Science, University of California at Davis. These references were also used for the identification of haemoglobin types of wild sheep. Frequencies of haemoglobin alleles were calculated according to Nix et al. (1969) and Lauvergne ( 1989):
EVOLUTIONARY IMPLICATIONSOF HAEMOGLOBIN POLYMORPHISM IN SHEEP
317
f = 2AA-t-.AB/ 2 (No. of animals sampled ) where AA and AB are the numbers of haemoglobin phenotypes HbflAHbflA and HbflAHbflB, respectively. The Chi-square method was used to test significant differences in allelic frequencies between sheep populations and the deviation from Hardy-Weinberg equilibrium. RESULTS
Phenotypic and allelic frequencies for the populations of wild and domesticated sheep sampled are in Table 1. Both Hbfl A and Hbfl B alleles were observed in nearly all domesticated sheep populations. The HbflB gene was more frequent than the HbflA gene in most cases except in Romanov, Finnsheep TABLE 1 Frequency of alleles at the haemoglobin (Hbfl) locus in domesticated and wild sheep in USA 1 Populations
Symbols
Phenotypes
Gene frequencies
N
AA
AB
BB
Hbflg
Hbfl a
Texas Nebraska Dubois, Idaho Nebraska Utah Texas Utah Dubois, Idaho Nebraska Northern Utah USVirgin Islands Southern Idaho Northern Utah Dubois, Idaho
20 40 35 38 41 20 30 34 30 35 30 30 31 33
0 13 1 14 1 0 0 0 23 8 7 0 0 1
2 18 14 18 9 5 1 5 7 19 8 0 9 7
18 9 20 6 31 15 29 29 0 8 15 30 22 25
0.05 0.55 0.23 0.60 0.13 0.12 0.02 0.07 0.88 0.50 0.37 0.00 0.15 0.14 0.29
0.95 0.45 0.77 0.40 0.87 0.88 0.98 0.93 0.12 0.50 0.63 1.00 0.85 0.86 0.71
AGMO
Utah
33
0
0
33
0.00
1.00
ARMM
Texas
28
0
0
28
0.00
1.00
DBIG
Utah and Arizona
31
0
0
31
0.00
1.00
Domesticated Sheep Barbados BARB Booroola BOOR Columbia COLM Finnsheel9 FINN Hampshi:re HAMP Karakul KARA Rambouillet- 1 RAMB 1 Rambouillet-2 RAMB2 Romano~ ROMA St. Croix- 1 STCX 1 St. Croix-2 STCX2 Suffolk- 1 SUFK1 Suffolk-2 SUFK2 Targhee TARG Pooled average frequencies. Wild Sheep Argali × European Mouflon Asiatic Mouflon × European Mouflon Desert Bighorn
Location
ISignificance of differences explained in text.
318
S. W A N G ET AL
<
o.
o ~D <
c~c~
{D < [..
a~ od a~
0
o
< <
©
< II
~j
o
A
z v
~,
f',l ..1 <
o "~
..~ 0 (D
"
0
~
g
v~
8~
EVOLUTIONARY IMPLICATIONS OF HAEMOGLOBIN POLYMORPHISM IN SHEEP
319
and Booroola. Average frequency of the Hbfl a allele for domesticated sheep was 0.71. The Hbfl B gene frequencies among populations of domesticated sheep fluctuated between 0.12 and 1.00. All wild sheep sampled in this survey were monomorphic for Hbfl B. One significant ( P < 0.05 ) deviation from the Hardy-Weinberg equilibrium was observed in St. Croix flock 2. This deviation was due to the lack of heterozygotes. There were no predictable relationships between breeds in the allelic frequencies of haemoglobin. Distantly related breeds may share the same types of alleles with similar frequencies, such as the Karakul compared to Targhee and Suffolk, and the Booroola to St. Croix (Table 1 ), while breeds sharing close relationship in origin and development may differ significantly ( P < 0.05 ), such as the Columbia compared to Rambouillet (Tables 1 and 2 ). Significant differences (Table 2) existed in the Hb gene distribution between Barbados vs. Finnsheep, Romanov vs. all other sheep populations ( P < 0.01 ), St. Croix vs. Rambouillet and Suffolk ( P < 0.01 ), and Targhee vs. Booroola ( P < 0 . 0 1 ) . Significant differences ( P < 0.05 ) were also observed between populations (flocks) of the Suffolk breed but not between those of St. Croix and Rambouillet breeds (Table 2 ). DISCUSSION
Nadler et al. ( 1971 ) reported that the wild sheep from Asia (Iran), Europe and North America (Ovis dalli, Ovis musimon and Ovis canadensis) were all Hbfl B type. Lay et al. ( 1971 ) and Bunch and Foote (1976) observed Hbfl a monomorphism in wild Iranian sheep (Ovis orientalis, Ovis vignei and the hybrids of the two). The Hbfl B was the only allele in wild sheep (Ovis ammon× Ovis musimon, Ovis orientalis× Ovis musimon and Ovis canadensis) observed in this study and confirms what has been previously reported. Agar et al. (1972) reviewed the Hb polymorphic distribution of sheep in many locations over the world, and reported that more than two thirds of the populations surveyed were more frequent in Hbfl B than Hbfl A. This was also observed in indigenous domestic breeds (Tan sheep and Mongolian sheep) in China (Wang, 1983, personal communication), and the major domestic sheep breeds in Iran (Bunch and Foote, 1976), the Nellore sheep in India (Reddy et al., 1988 ), the Askania and Tsigai sheep in USSR (Iovenko, 1987 ) and other native sheep breeds in Europe (Rizzi et al., 1988 ). Lay et al. ( 1971 ), Bunch et al. (1978) and Valdez et al. (1978) hypothesized that wild sheep gave rise to the Hbfl A allele, which most likely evolved after the domestication of sheep. Evidence for this conclusion was based upon the fact that the Hbfl A allele was not found in wild sheep and Hbfl B was more frequent than Hbfl g in most domesticated sheep. The molecular structure of haemoglobin seems to support this hypothesis.
320
s. WANG ETAL.
The Hbfl a of wild sheep is electrophoretically similar to Hbfl B of domesticated sheep. However, wild sheep Hbfl B differs from domesticated sheep Hbfl B (beta-chain) at five amino acids and from domesticated sheep Hbfl A only at two amino acids. These different amino acid positions do not overlap with each other (Manwell and Baker, 1976). According to the constancy of the amino acid substitution rate (Kimura, 1983 ), Hbfl A must have occurred later than Hbfl B, since it involved less amino acid substitutions. It is likely that the Hbfl A allele is now in the process of spreading throughout all breeds of domesticated sheep. The extensive variation of the haemoglobin polymorphic distribution in domesticated animals may be attributed to natural selection under different environments, differential selection and management and genetic drift. From our data we could not determine their relative influence. However, the rand o m pattern of allelic distribution might indicate that genetic drift is an important factor in causing variation. As shown by the results, relationships between sheep breeds in haemoglobin distribution in terms of alleles and their frequencies are not predictable (Table 1 ). Furthermore, flocks within the same breed may or may not show significant differences in allelic frequencies, and this seems not to be related to the variation in environments (Tables 1 and 2). Significant differences occurred between flocks of the same breed located in the same geographical region (two flocks of Suffolk, one from Northern Utah and the other from Southern Idaho). However, there was no significant difference between the two flocks of St. Croix and between the two flocks of Rambouillet. These two flocks in each breed were located in areas where the environments were quite different. One flock from the St. Croix breed (STCX 1 ) was located in Northern Utah. The flock was established in 1975 and propagated from two rams and 22 ewes from the Island of St. Croix in the Caribbean. The other St. Croix flock (STCX2) was located on St. Croix Islands and some of the animals are from the source flocks for STCX 1. Haemoglobin polymorphic distribution among flocks did not differ from each other (Table 1 ). CONCLUSION
The observation that allelic frequency ofHbfl B was greater than for the HbflA allele in most domesticated sheep breeds and was the only allele that existed in genetic groups of wild sheep, supports the hypothesis that the Hbfl A allele arose after domestication of sheep. The wide variation in allelic distribution among domesticated sheep populations most likely resulted from selection and genetic drift. The random pattern of haemoglobin polymorphic distribution indicates that genetic drift is an important factor leading to the polymorphic frequency differentiation.
EVOLUTIONARY IMPLICATIONS OF HAEMOGLOBIN POLYMORPH1SM IN SHEEP
321
ACKNOWLEDGMENTS W e are :grateful to Drs. C . M . T . P e n e d o , R. S w e n s o n , D. M a t t h e w s , S. Wildeus, K. E r c a n b r a c k , L. Y o u n g a n d M. S h e l t o n for p r o v i d i n g h a e m o g l o b i n s a m p l e s fi~r this study. Special t h a n k s to Mrs. A. M a c i u l i s for l a b o r a t o r y ass i s t a n c e a n d to M r . R . C . E v a n s for h e l p in collecting s a m p l e s . T h i s p a p e r is p u b l i s h e d as U t a h A g r i c u l t u r a l E x p e r i m e n t S t a t i o n J o u r n a l p a p e r n u m b e r 3840.
REFERENCES Andrew, A.T., 1986. Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications, 2nd Edn., Clarendon Press, Oxford, pp. 79-92. Agar, N.S., Evans, J.V. and Roberts, J., 1972. Red blood cell potassium and haemoglobin polymorphisrn in sheep. Anim. Breeding Abstr. 40 (3): 407-436. Baker, C.M.A. and Manwell, C., 1983. Electrophoretic variation of erythrocyte enzymes of domesticated mammals. In: N.S. Agar and P.G. Board (Editors), Red Blood Cells of Domestic Mammals, Elsevier North-Holland, Amsterdam, pp. 367-412. Blunt, M.H. and Huisman, T.H.J., 1975. The hemoglobin of sheep. In: M.H. Blunt (Editor), The Blood of Sheep, Springer-Verlag, New York, pp. 155-183. Buis, R.C. and Tucker, E.M., 1983. Relationships between rare breeds of sheep in Netherlands as based on blood typing. Anim. Blood Groups Biochem. Gen. 14:17-26. Bunch, T.D. and Foote, W.C., 1976. Chromosomes, hemoglobin, and transferrins of Iranian domesti,z sheep. J. Hered. 67:167-170. Bunch, T.D., Nguyen, T.C. and Lauvergne, J.J., 1978. Hemoglobin of the Corsica-Sardinian Mouflon (Ovis musimon) and their implications for the origin of HbA in domestic sheep (Ovis aries). Ann. Genet. Sel. Anim. 10: 503-506. Iovenko, V N., 1987. Comparison of the genetic structures of sheep populations using biochemical polymorphisms. Biol. 12: 35-39. (Anim. Breed. Abstr. 57: 1684). Kimura, M., 1983. The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. Lauvergne, J.J. (ed.), 1989. Standardized genetic nomenclature for sheep and goats 1987: Loci for visible traits other than colour and blood and milk polymorphisms. Proc. COGNOSAG Workshop July 21-26, 1987, Manosque, Bureau des Ressources Genetiques, Paris, pp. 184. Lay, D.M., Nadler, C.F. and Hassinger, J.D., 1971. The transferrins and hemoglobin of wild Iranian sheep. Comp. Biochem. Physiol. 40B: 521-529. Manwell, C. and Baker, C.M.A., 1976. Protein polymorphisms in domesticated species: Evidence for hybrid origin? In: S. Karlin and E. Nevo (Editors), Population Genetics and Ecology, Academic Press, New York, NY, USA, pp. 105-130. Manwell, C. and Baker, C.M.A., 1977. Genetic distance between the Australian Merino and the Poll Dorset sheep. Genet. Res. 29: 239-253. Nadler, C.F., Woolf, A. and Harris, K.E., 1971. The transferrins and hemoglobin of Bighorn sheep ( Ovis canadensis), Dall sheep ( Ovis dalli), and Mouflon ( Ovis musimon). Comp. Biochem. Physiol. 40B: 567-570. Nix, C.E., Bogart, R. and Price, D.A., 1969. Genetics of plasma transferrins in five breeds of Sheep. J. Hered. 60: 97-100. Reddy, V.P., Reddy, V.R.C. and Reddy, K.K., 1988. Genetics of haemoglobin types and their
322
s. WANG ET AL.
influence on growth traits in Nellore and Nellore X Dorset half bred sheep. Ind. J. Anim. Sci. 58: 1234-1236. Rizzi, R., Caroli, A., Bolla, P., Acciaioli, A. and Pagnacco, G., 1988. Variability of reduced glutathione levels in Masseses ewes and its effect on daily milk production. J. Dairy Res. 55: 345-353. Valdez, R., Nadler, C.F. and Bunch, T.D., 1978. Evolution of wild sheep in Iran. Evolution 32: 56-72. RESUMEN Wang, S., Foote, W.C. y Bunch, T.D., 1991. Implicationes evolutionair del polimorfismo en la hemoglobin en razas dom6sticas y salvajes ovejas. Small Rumin. Res., 4:315-322. La distribuci6n del polimorfismo en la hemoglobina fue investigado en 11 razas dom6sticas y tres grupos gen6ticos de ovejas saivajes en los Estados Unidos de Norteam6rica. Hbfl Bfue mas frecuente que HbflA en la mayoria de las razas dom6sticas con frecuencia promedio de 0.71. Las frecuencias del gen HbflB fueron: 0.95 (Barbados), 0.45 (Booroola), 0.77 (Columbia), 0.40 (Finnsheep), 0.87 (Hampshire), 0.88 (Karakul), 0.98 (Rambouillet flock 1), 0.93 (Rambouillet flock 2), 0.12 (Romanov), 0.50 (St. Croix flock 1), 0.63 (St. Croix flock 2), 1.00 (Suffolk flock 1 ), 0.86 (Suffolk flock 2) y 0.86 (Targhee). Todas las ovejas salvajes examinadas [Cruza de Argali X Mouflon Europea (Ovis ammon X Ovis musimon), cruza de Mouflon Asiatica × Mouflon Europea (Ovis orientalis × Ovis musimon) y Desert Bighorn (Ovis canadensis) ] fueron monom6rficas para HbflB. El monomorfismo de HbflBen ovejas salvajes y la alta frecuencia de Hbfl B comparada con HbflA en ovejas dom6sticas indica que HbflA probablemente se present6 despu6s de la domesticaci6n de la oveja. La amplia variaci6n del polimorfismo en la hemoglobina dentro de las razas y entre las razas puede haber sido consecuencia de una combinaci6n de migraci6n gen6tica al azar y selecci6n.