Animal Genetics 1992,23,275-278
SHORT COMMUNICATION
Demonstration of serum albumin (ALB) polymorphism in wild rabbits, Oryctolagus cuniculus, by means of isoelectric focusing N. FERRAND & J. ROCHA* Instituto de Zoologia, and *Institute de Antropologia, Faculdade de Ci&ncias, Universidade d o Porto, Porto, Portugal
Summary. Genetic polymorphism of serum albumin was demonstrated by isoelectric focusing in wild rabbit populations from Portugal and England. Gene frequencies were estimated to be (1)ALB.I = 0-47,ALB*2 = 0*49,ALB*3= 0.04, in Portugal, and (2) ALB*l = 0-60 and ALB*2 = 0-40, in England. One hundred Portuguese domestic rabbits of mixed breeds were all of ALB I type. Keywords: rabbit, albumin, genetic polymorphism, isoelectric focusing
To our knowledge, only a few reports concerning the electrophoretic behaviour of rabbit, Oryctolagus cuniculus, serum albumin (ALB) have been published up to now. These included the analysis of ALB by both one- and two-dimensional gel electrophoresis, using domestic breeds as a source of plasma samples, and failed to reveal any variation (Binette et a f . 1965; Zaragoza et al. 1984; Juneja et al. 1984). In this study, we reassess the problem of ALB variation in the rabbit, extending the investigation to wild populations, and using an isoelectric focusing (IEF) technique recently described (Rocha et al. 1991). We found evidence of a genetic polymorphism of ALB in wild populations from Portugal and England. Screening of Portuguese domestic rabbits did not reveal any variation at the ALB locus. Blood samples from wild rabbit populations were collected and treated as described in Ferrand et al. (1988), and from domestic animals of mixed breed origin (100 unrelated rabbits) according to Ferrand (1989). IEF was performed according to Rocha et a f . (1991). Briefly, a solution containing polyacrylamide (T = 5 % , C = 3%), 60mM L-serine, 8 M urea and 6.25% (v/v) carrier ampholytes (1:l mixture of pH 5-6 and pH 5-8, Pharmacia) was poured in a 235 X 100 x 0.3mm cassette. Gel polymerization was achieved in 1 h at room temperature by adding 0.7% (v/v) of a 0 . 4 4 ammonium ~ persulphate solution and 0.1% (vh) of TEMED. 0 . 0 4 aspartic ~
Correspondence: Nuno Ferrand, Instituto de Zoologia, Faculdade de Cihcias, Universidade do Porto, 4000 Porto, Portugal. Accepted 19 August 1991
275
276 N . Ferrand & J . Rocha
Figure 1. Patterns of the six common albumin phenotypes observed in wild rabbit populations after isoelectric focusing in the presence of 8hl urea and 6 0 m m L-serine.
acid and l i v l NaOH solutions were used for the anode and cathode electrodes, respectively. Gels were prefocused for 1h at 1SOOV and 2SmA with power limits being fixed at 0.7 W (30 min), 2 W (1.5min), and 3 W (15 min). After prefocusing, 7 pi of serum samples diluted 1:50 in distilled water was applied using a silicon rubber application strip (DESAGA) at 1cm from the cathode. Focusing was performed with the same voltage and current settings of prefocusing and the power limits were fixed at 3 W (3 h), 3.6 W (30 min) and 4.2 W (30 min). After focusing the gels were placed in a 10% (w/v) solution of trichloroacetic acid for 10 min. For protein staining a 0.1% solution of Kenacid Blue R (British Drug House) in ethanol-water (1:2, v/v) was employed. Destaining was performed with an ethanol-acetic acid-water (3:1:8, v/v) solution. Gene frequencies were estimated by direct gene counting. Figure 1 depicts six different patterns detected in wild rabbit after protein separation by IEF under the conditions described above. Identification of such patterns as albumin phenotypes was confirmed by immunofixation using undiluted goat anti-human albumin (ATAB). These phenotypes were interpreted as being determined by three alleles, A L B * I , ALB*2 and ALB*3, at an autosomal locus. Patterns designated as 1 . 2 and 3 represent the homozygotes for the A L B * l , ALB.2 and ALB*3 gene products, respectively. Patterns 2-1, 3-1 and 3-2 represent the heterozygotes and show the positions of each of the corresponding gene products. Table 1 shows the distribution of ALB phenotypes in two wild rabbit populations (most of Continental Portugal and Bridgets Farm, Winchester, England), and in a sample of Portuguese domestic rabbit of mixed breed origin. In this sample, ALB*Z was the only detected gene product. In the Portuguese wild population three common alleles were found, while in the English sample only two (ALB*I and A L B * 2 ) were observed. In both wild populations good agreement was found between observed and expected results on the basis of Hardy-Weinberg equilibrium.
Albumin polymorphism in rabbits 277
Table 1. Albumin gene frequencies in some wild and domestic rabbits. The numbers in parenthesis are expected values
Phenotypes Sample Wild rabbit Portugal ( 130) England
(100) Domestic rabbit
Gene frequencies
1
2-1
2
3-1
3-2
3
ALB*l
ALB*2
ALB*3
32 (28.7) 35 (36.0) 100
55
33 (31.2) 14 (164) -
4 (4.9) -
5 (5.1)
0.49
0.04
-
1 (0.2) -
0.47
(59.9) 51 (48.0)
0.60
0.40
-
-
-
-
1.00
-
-
-
(100)
The difficulties of performing controlled matings with wild rabbits prevent the presentation of family data. The data obtained from screening of Portuguese domestic rabbits confirmed previous results on the absence of variation at this locus in several domestic breeds (Binette et al. 1965; Zaragoza et al. 1984; Juneja et al. 1984). On the contrary, the analysis of wild populations, both from Portugal and England, reveals a high degree of genetic diversity of ALB. These findings indicate that this polymorphism can be particularly informative in the distinction of wild and domestic rabbit populations, and show the same trend as those obtained for transferrin polymorphism, which also occurs only in wild populations (Arana et al. 1987; Ferrand et al. 1988). However, a more detailed study is needed, since two recent investigations (Arana et al. 1989; Peterka et al. 1990) using 8 and 11 polymorphisms, respectively, were not able to distinguish clearly wild and domestic populations. This study, including the extension of both the number of genetic markers and sampled populations, is now in progress in our laboratories. Finally, we must stress that this IEF technique seems to constitute a sensitive and reliable method that may prove to be useful for the investigation of genetic variation of ALB in other species besides rabbit.
Acknowledgments The authors are very grateful to Paul Sunnucks for allowing the usage of his sera collected in the English wild rabbit population, and to the anonymous referees whose criticisms considerably improved the content of this paper. Part of this work was presented at the 22nd International Conference on Animal Genetics, East Lansing, Michigan, USA, 1990.
References Arana Z., Zaragoza P., Rodellar C. & Arnorena B. (1987) Evidence for transferrin polymorphism in Spanish wild rabbits. Animal Genetics 18, 125-32.
278 N . Ferrand & J . Rocha
.4rana A., Zaragoza P., Rodellar C. & Amorena B. (1989) Blood biochemical polymorphisms as markers for genetic characteristics of wild Spanish and domestic rabbits. Genefica 79, 1-9. Binette J.P., MacNair M.B. & Calkins E. (1965) Fractionation and characterization of normal rabbit plasma proteins. Biochemica( Journal 94, 143. Ferrand N . (1989) Biochemical and genetic studies on rabbit hemoglobin. Electrophoretic polymorphism of the P-chain. Biochemical Genetics 27, 673-8. Ferrand N., Carvalho G . & Amorim A. (1988) Transferrin (Tf) polymorphism in wild rabbit, Oryctolagus cuniculus. Animal Genetics 19, 295-300. Juneja R.K., Van de Weghe A. & Gahne B. (1984) Genetic polymorphism of serum postalbumin (Po) and pretransferrin (Prt) in the laboratory rabbit. Hereditus 100, 11-15. Peterka M., Hart1 G.B. & Hoger H. (1990) Genetic diversity within and between populations of wild and domestic rabbits (Oryctolagus cuniculus). 2nd Symposium on Genetics of Wild Animals, Giessen, March 1990. Rocha J., Kompf J . , Ferrand N . , Amorim A. & Ritter H. (1991) Separation of human alloalbumin variants by isoelectric focusing. Electrophoresis 12, 31S14. Zaragoza M.P., Amorena B., Arana A. & Zarazaga I. (1984) Advances metodoldgicos para la deteccidn de variaciones electroforkticas e n suer0 du conejo. Anales de la Facultad Veferinaria 18/19, 183-96.
SHORT COMMUNICATION
Demonstration of serum albumin (ALB) polymorphism in wild rabbits, Oryctolagus cuniculus, by means of isoelectric focusing N. FERRAND & J. ROCHA* Instituto de Zoologia, and *Institute de Antropologia, Faculdade de Ci&ncias, Universidade d o Porto, Porto, Portugal
Summary. Genetic polymorphism of serum albumin was demonstrated by isoelectric focusing in wild rabbit populations from Portugal and England. Gene frequencies were estimated to be (1)ALB.I = 0-47,ALB*2 = 0*49,ALB*3= 0.04, in Portugal, and (2) ALB*l = 0-60 and ALB*2 = 0-40, in England. One hundred Portuguese domestic rabbits of mixed breeds were all of ALB I type. Keywords: rabbit, albumin, genetic polymorphism, isoelectric focusing
To our knowledge, only a few reports concerning the electrophoretic behaviour of rabbit, Oryctolagus cuniculus, serum albumin (ALB) have been published up to now. These included the analysis of ALB by both one- and two-dimensional gel electrophoresis, using domestic breeds as a source of plasma samples, and failed to reveal any variation (Binette et a f . 1965; Zaragoza et al. 1984; Juneja et al. 1984). In this study, we reassess the problem of ALB variation in the rabbit, extending the investigation to wild populations, and using an isoelectric focusing (IEF) technique recently described (Rocha et al. 1991). We found evidence of a genetic polymorphism of ALB in wild populations from Portugal and England. Screening of Portuguese domestic rabbits did not reveal any variation at the ALB locus. Blood samples from wild rabbit populations were collected and treated as described in Ferrand et al. (1988), and from domestic animals of mixed breed origin (100 unrelated rabbits) according to Ferrand (1989). IEF was performed according to Rocha et a f . (1991). Briefly, a solution containing polyacrylamide (T = 5 % , C = 3%), 60mM L-serine, 8 M urea and 6.25% (v/v) carrier ampholytes (1:l mixture of pH 5-6 and pH 5-8, Pharmacia) was poured in a 235 X 100 x 0.3mm cassette. Gel polymerization was achieved in 1 h at room temperature by adding 0.7% (v/v) of a 0 . 4 4 ammonium ~ persulphate solution and 0.1% (vh) of TEMED. 0 . 0 4 aspartic ~
Correspondence: Nuno Ferrand, Instituto de Zoologia, Faculdade de Cihcias, Universidade do Porto, 4000 Porto, Portugal. Accepted 19 August 1991
275
276 N . Ferrand & J . Rocha
Figure 1. Patterns of the six common albumin phenotypes observed in wild rabbit populations after isoelectric focusing in the presence of 8hl urea and 6 0 m m L-serine.
acid and l i v l NaOH solutions were used for the anode and cathode electrodes, respectively. Gels were prefocused for 1h at 1SOOV and 2SmA with power limits being fixed at 0.7 W (30 min), 2 W (1.5min), and 3 W (15 min). After prefocusing, 7 pi of serum samples diluted 1:50 in distilled water was applied using a silicon rubber application strip (DESAGA) at 1cm from the cathode. Focusing was performed with the same voltage and current settings of prefocusing and the power limits were fixed at 3 W (3 h), 3.6 W (30 min) and 4.2 W (30 min). After focusing the gels were placed in a 10% (w/v) solution of trichloroacetic acid for 10 min. For protein staining a 0.1% solution of Kenacid Blue R (British Drug House) in ethanol-water (1:2, v/v) was employed. Destaining was performed with an ethanol-acetic acid-water (3:1:8, v/v) solution. Gene frequencies were estimated by direct gene counting. Figure 1 depicts six different patterns detected in wild rabbit after protein separation by IEF under the conditions described above. Identification of such patterns as albumin phenotypes was confirmed by immunofixation using undiluted goat anti-human albumin (ATAB). These phenotypes were interpreted as being determined by three alleles, A L B * I , ALB*2 and ALB*3, at an autosomal locus. Patterns designated as 1 . 2 and 3 represent the homozygotes for the A L B * l , ALB.2 and ALB*3 gene products, respectively. Patterns 2-1, 3-1 and 3-2 represent the heterozygotes and show the positions of each of the corresponding gene products. Table 1 shows the distribution of ALB phenotypes in two wild rabbit populations (most of Continental Portugal and Bridgets Farm, Winchester, England), and in a sample of Portuguese domestic rabbit of mixed breed origin. In this sample, ALB*Z was the only detected gene product. In the Portuguese wild population three common alleles were found, while in the English sample only two (ALB*I and A L B * 2 ) were observed. In both wild populations good agreement was found between observed and expected results on the basis of Hardy-Weinberg equilibrium.
Albumin polymorphism in rabbits 277
Table 1. Albumin gene frequencies in some wild and domestic rabbits. The numbers in parenthesis are expected values
Phenotypes Sample Wild rabbit Portugal ( 130) England
(100) Domestic rabbit
Gene frequencies
1
2-1
2
3-1
3-2
3
ALB*l
ALB*2
ALB*3
32 (28.7) 35 (36.0) 100
55
33 (31.2) 14 (164) -
4 (4.9) -
5 (5.1)
0.49
0.04
-
1 (0.2) -
0.47
(59.9) 51 (48.0)
0.60
0.40
-
-
-
-
1.00
-
-
-
(100)
The difficulties of performing controlled matings with wild rabbits prevent the presentation of family data. The data obtained from screening of Portuguese domestic rabbits confirmed previous results on the absence of variation at this locus in several domestic breeds (Binette et al. 1965; Zaragoza et al. 1984; Juneja et al. 1984). On the contrary, the analysis of wild populations, both from Portugal and England, reveals a high degree of genetic diversity of ALB. These findings indicate that this polymorphism can be particularly informative in the distinction of wild and domestic rabbit populations, and show the same trend as those obtained for transferrin polymorphism, which also occurs only in wild populations (Arana et al. 1987; Ferrand et al. 1988). However, a more detailed study is needed, since two recent investigations (Arana et al. 1989; Peterka et al. 1990) using 8 and 11 polymorphisms, respectively, were not able to distinguish clearly wild and domestic populations. This study, including the extension of both the number of genetic markers and sampled populations, is now in progress in our laboratories. Finally, we must stress that this IEF technique seems to constitute a sensitive and reliable method that may prove to be useful for the investigation of genetic variation of ALB in other species besides rabbit.
Acknowledgments The authors are very grateful to Paul Sunnucks for allowing the usage of his sera collected in the English wild rabbit population, and to the anonymous referees whose criticisms considerably improved the content of this paper. Part of this work was presented at the 22nd International Conference on Animal Genetics, East Lansing, Michigan, USA, 1990.
References Arana Z., Zaragoza P., Rodellar C. & Arnorena B. (1987) Evidence for transferrin polymorphism in Spanish wild rabbits. Animal Genetics 18, 125-32.
278 N . Ferrand & J . Rocha
.4rana A., Zaragoza P., Rodellar C. & Amorena B. (1989) Blood biochemical polymorphisms as markers for genetic characteristics of wild Spanish and domestic rabbits. Genefica 79, 1-9. Binette J.P., MacNair M.B. & Calkins E. (1965) Fractionation and characterization of normal rabbit plasma proteins. Biochemica( Journal 94, 143. Ferrand N . (1989) Biochemical and genetic studies on rabbit hemoglobin. Electrophoretic polymorphism of the P-chain. Biochemical Genetics 27, 673-8. Ferrand N., Carvalho G . & Amorim A. (1988) Transferrin (Tf) polymorphism in wild rabbit, Oryctolagus cuniculus. Animal Genetics 19, 295-300. Juneja R.K., Van de Weghe A. & Gahne B. (1984) Genetic polymorphism of serum postalbumin (Po) and pretransferrin (Prt) in the laboratory rabbit. Hereditus 100, 11-15. Peterka M., Hart1 G.B. & Hoger H. (1990) Genetic diversity within and between populations of wild and domestic rabbits (Oryctolagus cuniculus). 2nd Symposium on Genetics of Wild Animals, Giessen, March 1990. Rocha J., Kompf J . , Ferrand N . , Amorim A. & Ritter H. (1991) Separation of human alloalbumin variants by isoelectric focusing. Electrophoresis 12, 31S14. Zaragoza M.P., Amorena B., Arana A. & Zarazaga I. (1984) Advances metodoldgicos para la deteccidn de variaciones electroforkticas e n suer0 du conejo. Anales de la Facultad Veferinaria 18/19, 183-96.
