Protein polymorphism in native goats from central Argentina
C. Deza
a, G.T. PeÂrez
a, C.N. Gardenal
b, L. Varela
a, M. Villar
a,
S. Rubiales
c, C. Barioglio
a,*aFacultad de Ciencias Agropecuarias, Universidad Nacional de CoÂrdoba, Casilla de Correo 509,
5000 CoÂrdoba, Argentina
bCaÂtedra de QuõÂmica BioloÂgica, Facultad de Ciencias MeÂdicas, Universidad Nacional de CoÂrdoba,
Casilla de Correo 35 Sucursal 16, 5000 CoÂrdoba, Argentina
cCaÂtedra de FisiologõÂa Humana, Facultad de Ciencias QuõÂmicas, Universidad Nacional de CoÂrdoba,
Casilla de Correo 61, 5000 CoÂrdoba, Argentina
Accepted 15 July 1999
Abstract
Under the name of Creole goats, individuals of various origins, morphological and productivity characters are grouped. To characterize the genetic pool of goats from different sources in central Argentina, protein polymorphism revealed by electrophoresis was analyzed. A total of 109 adult animals from Santa MarõÂa, ColoÂn and IschilõÂn Districts, CoÂrdoba Province, were studied. The red cells lysates were subjected to starch gel electrophoresis and speci®c staining procedures to reveal the activity of different enzymes and hemoglobin. Separation of plasmatic proteins was carried out by electrophoresis in polyacrylamide gel and stained with Coomassie Blue. From a total of 14 loci analyzed, eight were polymorphic in at least one ¯ock. Four codominant alleles were detected in the locusbHemoglobin (bHb); three alleles in malic enzyme (Me), glucose 6 phosphate dehydrogenase (G6pdh) and lactate dehydrogenase (Ldh) and two alleles in catalase (Cat), transferrin (Tf), esterase-2 (Es-2) and phosphoglucomutase (Pgm). In all the cases, the observed genotype frequencies were not signi®cantly different from those expected from Hardy±Weinberg equilibrium. The proportion of polymorphic loci (P%) varied between 21.4% and 42.9% and mean heterozygosity (H), between 0.061 and 0.117. Alleles at loci Cat, Me and Es-2 showed signi®cantly different frequencies according to the sample origin. The ¯ock from ColoÂn District presented the highest levels of polymorphism. This is the only stock which includes hybrids of varying grade between Creole stocks and male specimens from Saanen and Nubian breeds.#2000 Published by Elsevier Science B.V. All rights reserved.
Keywords:Protein polymorphism; Creole goats; Biochemical markers
1. Introduction
Among farm animals, relatively few data on bio-chemical polymorphism have been reported in goats.
Studies of genetic variability using blood markers were performed in breeds from Europe, South Africa and Japan (Nozawa et al., 1978; Katsumata et al., 1981; Osterhoff et al., 1987; TunÄon et al., 1989; Menrad et al., 1994; Rodero et al., 1997), but there is no information on protein polymorphism in native goats from South America.
*Corresponding author. Fax:54-51-334-118.
E-mail address: [email protected] (C. Barioglio).
The present goat population in Argentina is about 4,000,000 head, distributed mainly in arid and semi-arid regions of the country. Most of the animals are assigned to a not well de®ned type of goats described as straight cephalic pro®le with short ears and small sable horns known as Creole (Mason, 1981), resulting from centuries of uncontrolled breeding and natural selection from the original stocks introduced by the Spanish colonizers in the 16th century. Some authors (Agraz-GarcõÂa, 1976) maintain that these stocks derived from the Celtibera White breed, but Mason (1981) considers that they derived from the Granada, Murciana and Malaga Spanish breeds. Pure Saanen, Togenburg, Anglonubian and Angora were also intro-duced to improve meat and milk production in native stocks 40 years ago. Salinas and Dellepiane (1994) refer to the incorporation of pure Nubian animals (of African origin) by 1970. The degree of crossing of Creole goats from Argentina, with these breeds is poorly known. The objective of this study was to characterize the genetic pool of Creole goat ¯ocks from central Argentina through analyzing protein polymorphism in different herds.
2. Materials and methods
A total of 109 adult native goats from four different ¯ocks in four areas of CoÂrdoba Province (central Argentina) were sampled.
1. Sample 1: 34 female Creole goats, Farm of the School of Agronomy, National University of CoÂrdoba, Santa MarõÂa District.
2. Sample 2: 30 female Creole goats, Navarro`s farm, ColoÂn District. The present stock includes Creole animals and hybrid individuals of varying grades between native stocks and Saanen and Nubian, introduced with the aim of improving dairy production.
3. Sample 3: 45 female Creole goats, Bianchi`s farm, IschilõÂn District.
Blood samples were obtained from the jugular vein and placed in tubes with heparine as anticoagulant, refrigerated and carried to the laboratory. The red cells were separated, washed in saline solution and lysed with distillated water. Plasma and red cells were stored atÿ208C until tested.
The red cells lysates were submitted to starch gel electrophoresis as described by Smithies (1955) mod-i®ed by Boyer and Hiner (1963). Staining procedures for enzymes are those given by Shaw and Prasad (1970), and Harris and Hopkinson (1976). The enzymes analyzed were catalase (Cat), esterase (Es-1, Es-2), malic enzyme (Me), phosphoglucomutase (Pgm-1, Pgm-2), glucose phosphate isomerase (Gpi), glucose 6-phosphate dehydrogenase (G6pdh), lactate dehydrogenase (Ldh), isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh) andbhemoglobin (bHb). Electrophoretic methods are given in a Table 1. Globin
Table 1
Separation methods of proteins in blood from Creole goats
Protein Locus EC number System Electrode buffer Gel buffer
bHemoglobine bHb ± PAGEa Tris±glycine±8M urea pH 8.3 Tris±HCl±8M urea pH 8.6 Catalase Cat 1.11.1.6 STAGEb Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Malic enzyme Me 1.1.1.40 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Glucose 6-phosphate dehydrogenase G6pdh 1.1.1.49 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Phosphoglucomutase 1 and 2 Pgm-1 Pgm-2 5.4.2.2 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Lactate dehydrogenase Ldh 1.1.1.27 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Esterase 1 and 2 Es-1 Es-2 3.1.1.1 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Malate dehydrogenase Mdh 1.1.1.37 STAGE Tris±Citric, pH 6.3 Tris±Citric, pH 6.3d
Isocitrate dehydrogenase Idh 1.1.1.42 STAGE Tris±Citric, pH 6.3 Tris±Citric pH 6.3d
Glucose phosphate isomerase Gpi 5.3.1.9 STAGE Tris±Borate±EDTA, pH 8.6 Tris±Borate±EDTAc, pH 8.6
Transferrin Tf ± PAGE Tris±glycine pH 8.3 Tris±HCl pH 8.6
Albumin Al ± PAGE Tris±glycine pH 8.3 Tris±HCl pH 8.6
chains were dissociated by incubation of the red cells lysates in the presence of 8M urea and b -mercap-toethanol. Globin chains were separated by electro-phoresis in cellulose acetate, in buffer Tris±Boric± EDTA, pH 8.9, 8M urea (Hammemberg et al., 1974).
bglobin chains were separated by electrophoresis in polyacrylamide gel in presence of 8M urea. In both cases, Coomassie Blue R 250 was used in dying.
Separation of plasma proteins was carried out by electrophoresis in native polyacrylamide gel, with 7.5% of acrylamide concentration. Gels were stained with Coomassie Blue R 250. The loci analyzed were transferrin (Tf) and albumin (Al).
For molecular forms generated by multiple alleles, the one migrating faster toward the anode was desig-nated `A' and the following `B', `C', etc., in order of decreasing anodal mobility.
Allele frequencies and mean observed and expected heterozygosity estimates for each population were obtained using theBIOSYS-1 computer program (Swof-ford and Selander, 1989).
3. Results and discussion
Although the mode of inheritance of the protein variants observed was not proved by family segrega-tion, electrophoretic patterns obtained were similar to those described for other species of mammals where the genetic control was well established.
The enzymes and proteins analyzed gave informa-tion on a total of 14 loci. Eight of them were poly-morphic (Table 2). The loci Alb, Gpi, Idh, Mdh, Es-1 and Pgm-1 were monomorphic for the same allele in all the samples. The locus Alb was polymorphic in several Spanish breeds (Barbancho et al., 1980; Rodero et al., 1997) and Japanesse Saanen goats (Nozawa et al., 1978).
3.1. Hemoglobin
Starch electrophoresis of native Hb revealed four phenotypes (Fig. 1A). When dissociated hemoglobin chains were run in 8M urea on cellulose acetate,a
-Table 2
Allele frequencies for polymorphic loci in Creole goats
Locus Allele Sample
Santa MarõÂa (n34) ColoÂn (n30) IschilõÂn (n45)
bHb A 0.02 0.00 0.00
B 0.98 0.88 0.86
C 0.00 0.02 0.04
D 0.00 0.10 0.10
Cat A 0.94 0.78 0.24
B 0.06 0.22 0.76
Me A 0.03 0.10 0.13
B 0.96 0.82 0.87
C 0.01 0.08 0.00
G6pdh A 0.07 0.04 0.07
B 0.93 0.94 0.93
C 0.00 0.02 0.00
Pgm-2 A 0.00 0.03 0.00
B 1.00 0.97 1.00
Ldh A 0.00 0.00 0.01
B 0.03 0.00 0.03
C 0.97 1.00 0.96
Es-2 A 1.00 0.93 1.00
B 0.00 0.07 0.00
Tf A 0.65 0.59 0.67
polypeptides migrate cathodally whileb-polypeptides migrate anodally.a-chains showed a single cathodic band in all different phenotypes of native Hb, whileb -chains showed polymorphism. To obtain better reso-lution in the electrophoretic patterns, the samples were run in polyacrylamide gels in presence of 8M urea. The phenotypes observed were equivalent to those obtained in starch gels and can be interpreted as the product of four codominant alleles at locusbHb. In all the ¯ocks allele B was clearly predominant, while allele A was present only in one specimen from Santa MarõÂa (sample 1, Table 2). For Saanen goats from Japan, Nozawa et al. (1978) also reported a mono-morphic aHb locus and four phenotypes in bHb, determined by three codominant alleles. In goats from Spain belonging to the breeds Negra Serrana (Rodero et al., 1992), Andalusian White, Andalusian Black (Rodero et al., 1997), Murciana, Granadina, Mala-guenÄa y Serrana Andaluza (Barbancho et al., 1980) and in the other native Spanish breeds (TunÄon et al., 1989; Garcia-Casas et al., 1992), two alleles were detected, being A the one with higher frequency in all the breeds. On the basis of its higher frequency, allele B in our samples (Table 2) could be equivalent to allele one of Japanese goats and allele A in Spanish stocks, but this point should be clari®ed by comparative
studies. The allele HbD ocurred in goats from ColoÂn and IschilõÂn District with a frequency of 0.1 (Table 2). In French Saanen goats this allele was detected with a frequency of 0.15 (Pepin and Nguyen, 1994).
3.2. Catalase
Three phenotypes were found, which were desig-nated A for the faster migrating band, B for the slower migrating band and AB for a wide zone including both bands (Fig. 1B). The three phenotypes in catalase might be explained by the presence of codominant alleles A and B, the last one being the predominant in samples 1 and 2. In Andalusian White and Black Spanish breeds also, two alleles were found, fast and slow; allele S is the one with higher frequency in both cases, 0.53 and 0.84 respectively (Rodero et al., 1997). Probably they are equivalent to our A and B alleles. Locus Cat was monomorphic in Saanen goats from Japan (Nozawa et al., 1978).
3.3. Malic enzyme
After electrophoretic separation, four different phe-notypes could be distinguished: A, AB, B and BC (Fig. 1C). Phenotype A appeared only in sample 3 (¯ock
from IschilõÂn). The wide banded patterns would cor-respond to heterozygote genotypes AB and BC, the latter was only found in goats from ColoÂn District (sample 2). Rasero et al. (1989) also found three alleles for this locus in three goat populations from Sicilia. As in our ¯ocks (Table 2), allele designated as B was the most common.
3.4. Glucose 6 phosphate dehydrogenase
Three phenotypic variants of G6pdh, named AB, B, BC, have been observed in this study (Fig. 1D). The heterozygote genotype BC was found in only three of the animals tested. In the all ¯ocks the predominance of allele B was observed (Table 2).
3.5. Phosphoglucomutase
Products of two different loci, Pgm-1 and Pgm-2, were observed for this enzyme. Pgm-1 was mono-morphic in all samples. In Pgm-2, two phenotypes were found, AB and B (Fig. 2A). The wider and more anodical not well de®ned band would correspond to heterozygote genotype AB which appears only in goats from ColoÂn District. In all the ¯ocks the
pre-dominance of allele B was observed, while allele A was present in goats from ColoÂn District exclusively (Table 2). Studies in goats of Improved Fawn and Boer showed no variation at this locus (Menrad et al., 1994).
3.6. Lactate dehydrogenase
After electrophoretic separation three phenotypes were observed (Fig. 2B). Patterns of homozygote genotypes presented a single anodical band. The other phenotypes observed would correspond to heterozy-gote AC (found only in goats from IschilõÂn) and BC, observed in goats from samples 2 and 3 (ColoÂn and IschilõÂn Districts respectively).
3.7. Esterase-2
Two zones of activity corresponding to different loci were observed (Es-1 and Es-2). Es-1 was mono-morphic and in Es-2, two different phenotypes were detected (Fig. 2C). Phenotype A was found in goats from all districts, while phenotype AB appeared only in the ¯ock of ColoÂn District.
The loci Ldh, G6pdh and Es-2 were monomorphic in Saanen goats from Japan (Nozawa et al., 1978); in
contrast results obtained in this study indicate that these loci are polymorphic in at least in one stock of Creole goats.
3.8. Transferrin
After electrophoretic separation of serum proteins, three Tf phenotypes were observed (Fig. 2D). Pheno-types with a single band correspond to homozygotes for allele A (fast) or B (slow), while the heterozygotes (AB) presented two bands. In improved Fawn and Boer breeds from Germany, three phenotypes were also found (Menrad et al., 1994), while in Spanish breeds (Murciana, Granadina, MalaguenÄa and Serrana Andaluza) four phenotypes were observed, deter-mined by three alleles: A, B and C, the latter having a very low frequency (GarzoÂn et al., 1976; Barbancho et al., 1980; Rodero et al., 1997). Osterhoff and Ward-Cox (1972) detected a Tf D allele in Boer goats from South Africa. Up to now, allele A has been reported as the most frequent in different breeds, except in Saanen from Japan (Watanabe, 1971), where allele B was the most common. In our stocks allele A was predominant in all samples. Notwithstanding, allele B had a fre-quency of 0.41 in the ¯ock from ColoÂn District; this is the only sample which includes descendants from crosses with Saanen breed.
3.9. Polymorphism estimation
Allele frequencies of the polymorphic loci for each ¯ock are given in Table 2. Observed phenotypic frequencies were not signi®cantly different from those expected from the Hardy±Weinberg equilibrium.
Table 3 presents mean observed and expected het-erozygosity values, proportion of polymorphic loci and mean number of alleles per locus.Alleles at loci
Cat, Me and Es-2 showed signi®cantly different fre-quencies according to the sample origin (2: 88.38,
df2, 2: 14.43, df4, p< 0.01 and 2: 7.78,
df2, p< 0.05 respectively). Although differences in locusbHb were not signi®cant, they were very close to the limit value (p0.051). Sample 2 (ColoÂn Dis-trict) showed in loci G6pdh, Pgm-2 and Es-2, alleles not present in the other populations, with a frequency higher than 1% (Table 2). In locus Cat, allele A was the most frequent in samples 1 and 2 while in sample 3, allele B was the most common. In samples 2 and 3, levels of polymorphism (Table 3) are much higher than those reported for goats from Okinawa Islands of Japan (27 loci; Nozawa et al., 1978) and lower than the values obtained by Rodero et al. (1997) in Andaluzian White and Black breeds, on the basis of only ®ve protein loci.
Sample 2 (ColoÂn District) showed the highest levels of polymorphism (P42.90%; H0.117). This
¯ock is the only one where males from Saanen and Nubian breeds have been introduced in the last years. However, the ¯ock from IschilõÂn, which at least phe-notipically can be characterized as Creole, presents a similarly high degree of polymorphism when com-pared to the sample from ColoÂn District. Further comparative studies of allele frequencies including pure breeds, could allow to assess possible origin and degree of hybridization of native goats in central Argentina.
4. Conclusion
This is the ®rst study on blood protein polymorph-ism in goats from Argentina. The eight polymorphic loci detected in samples from different ¯ocks would be valuable to evaluate the contribution of different Table 3
Levels of proteinic polymorphism in three ¯ocks of Creole goats
District Mean number of alleles per locus (A)
Percentage of polymorphic loci (P%)
Mean heterozygosis per locus (H)a
H0 He
Santa MarõÂa 1.50 (0.2) 21.40 0.059 (0.029) 0.061 (0.033)
ColoÂn 1.70 (0.2) 42.90 0.116 (0.040) 0.117 (0.042)
IschilõÂn 1.60 (0.2) 35.70 0.111 (0.043) 0.108 (0.042)
Mean 1.60 (0.2) 33.03 0.095 (0.040) 0.095 (0.042)
aObserved (H
breeds to the almost unknown genetic pool of Creole goats from Argentina.
Acknowledgements
We thank Mr. E. Navarro and Ing. Agr. M. Bianchi for giving us access to their herds; M. Arias for her co-operation in obtaining samples and M. Chiappero for her technical assistance in the laboratory. This work has been supported by grants from the Consejo de Investigaciones Cienti®cas y TecnoloÂgicas of CoÂrdoba Province (CONICOR) and Secretaria de Ciencia y TeÂcnica de la Universidad Nacional de CoÂrdoba (SECYT). CNG. is a Career Investigator of Consejo Nacional de Investigaciones Cienti®cas y TeÂcnicas of Argentina (CONICET).
References
Agraz-GarcõÂa, A., 1976. Estudio zootecnico de tres razas caprinas. Ed. Acribia. Zaragosa, EspanÄa, p. 211.
Barbancho, M., Llanes, D., Morera, L., GarzoÂn, R., Rodero, A., 1980. Biochemical polymorphisms in spanish goat breeds. I. Murciana, Granadina, MalaguenÄa, MalaguenÄa and Serrana Andaluza. Archivos de Zootecnia 29, 259±274.
Boyer, S., Hiner, H., 1963. Modi®ed apparatus for starch electrophoresis. J. Lab. Clin. Med. 61, 879±881.
Garcia-Casas, C., Moreno, A., Capote, J., de la Haba, M.R., 1992. Characterization of the canary racial goat groups with erythrocyte genetic markers. Small Rumin. Res. 7, 361±368. Gardenal, C.N., Blanco, A., 1985. Polimor®smo enzimaÂtico en
Calomys musculinus: nueva estimacioÂn. Mendeliana 7, 3±12. GarzoÂn, R., Zarazaga, I., Vallejo, M., Rodero, A., 1976.
Polimor®smo bioquõÂmico de la raza caprina granadina. Arch. Zootec. 25, 147±152.
Hammemberg, B., Brett, I., Kitche, H., 1974. Ontogeny of hemoglobins in sheep. Ann. N.Y. Acad. Sci. 241, 672±680. Harris, H., Hopkinson, D.A., 1976. Handbook of Enzyme
Electrophoresis in Human Genetics. North Holland, Amster-dam, 352 pp.
Katsumata, M., Nozawa, K., Amano, T., Shinjo, A., Abe, T., 1981. Blood protein gene constitution of the Japanese Saanen breed of goat. Japanese J. Zootec. Sci. 52, 553±561.
Markert, C.L., Faulhaber, I., 1965. Lactate dehydrogenase isozyme patterns of ®sh. J. Exp. Zool. 159, 319±325.
Mason, I.L., 1981. In: Gall, C. (Ed.), Breeds in Goat Production, ch. 3. Academic Press, New York, p. 619.
Menrad, M., MuÈller, E., Stier, C.H., Gelderman, H., Gall, C., 1994. Protein polymorphisms in the blood of German improved Fawn and Boer goats. Small Rumin. Res. 14, 49±54.
Nozawa, K., Shinjo, A., Shotake, T., 1978. Populations genetics of farm animals. III. blood proteins variation in the meet goats in Okinawa Islands of Japan. Z. TierzuÈcht. ZuÈchtgsbiol. 95, 60±77.
Osterhoff, D.R., Schmid, D.O., Schoeman, S.M., 1987. The stability of genetic markers as identi®ed in goats. South African J. Anim. Sci. 17, 133±137.
Osterhoff, D.R., Ward-Cox, I.S., 1972. Serum polymorphism in three South African goat breeds. Proc. 12th Eur. Conf. Anim. Blood Grps Biochem. Polymorphism, Budapest, Hungary, pp. 579±582.
Pepin, L., Nguyen, T.C., 1994. Blood groups and protein polymorphisms in ®ve goat breeds (Capra hircus). Anim. Genetics 25, 333±336.
Rasero, R., Di-Stasio, L., Giaccone, P., Facello, C., 1989. Malic enzime polymorphism in goats. Anim. Genetics. 20(1), 80. Rodero, E., de la Haba, M.R., Rodero, A., 1997. Genetic study of
Andalusia`s ovine and caprine breeds. J. Anim. Breed. Genet. 114, 143±161.
Rodero, E., de la Haba, M.R., Zamorano, M.J., Rodero, A., Gonzalez, A., 1992. Study of genetic variability of the Negra Serrana goat breed. Arch. Zootec. 41, 537±542.
Salinas, A., Dellepiane, G., 1994. SituacioÂn del sector caprino nacional. PublicacioÂn del Ministerio de EconomõÂa, Obras y Servicios PuÂblicos, Argentina, p. 35.
Shaw, C., Prasad, R., 1970. Starch gel electrophoresis of enzymes. A compilation of recipes. Biochem. Genet. 4, 297±320. Smithies, O., 1955. Zone electrophoresis in starch gels: Group
variations in the serum proteins of normal human adults. Biochem. 61, 629±641.
Swofford, D.L., Selander, R.B., 1989. BIOSYS-1, A Computer Program for the Analysis Allelic Variation in Population Genetics and Biochemical Systematic. Release 1.7, Illinois Natural History Survey, Campaign III, p. 43.
TunÄon, M.J., Gonzales, P., Vallejo, M., 1989. Genetic relationships between 14 native Spanish breeds of goat. Anim. Genetics 20, 205±212.
