MILK PROTEIN POLYMORPHISMS AS GENETIC MARKER IN KOREAN NATIVE CATTLE

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Introduction
Since the initial discovery of two variants of 8lactoglobulin (8・LG) in cow's milk by Aschaffenburg and Drewry (1955), genetically controlled variation of all five m^or milk proteins has been demonstrated (Eigel et al., 1984).Until now the fliSj-casein (as广CN) locus has 5 alleles (A, B, C, D and E).^-casein (8-CN) locus shows 7 alleles (A1, A2, A3, B, C, D and E).x-casein (x-CN) has 4 alleles (A, B, C and E) and ^-lactoglobulin occurs in 9 different variants (A, B, C, D, E, F, G, H and W) and alactalbumin (q-LA) 아lows 3 alleles (A, B and C).These major milk proteins are controlled by codominant autosomal genes according to the Mendelian law of inheritance (Aschaffenburg, 1968).The various genetic variants differ from each other as a consequence of either substitution or deletion of amino acids within the polypeptide chain (Eigel et al., 1984).Charge differences in their protein molecules allow the separation and detection of the different variants by electrophoresis.
Thus, information is required on the gene frequencies of milk protein genetic variants in the population.The frequencies of marker genes which determine protein variants in cattle can also be used to indicate the ancestral origins and the genetic relationships between cattle breeds (Rendel, 1967;Kidd and Pircher, 1971;Kidd, 1974;Baker, 1982;Graml and Pirchner, 1984;Gonzalez et al., 1987).
The Korean native cattle (Bos taurus chosenus) are unique indigenous cattle breed raised in Korea* The origin of the Korean native ca비e is considered to be crossbred of Bos primigenius and Bos indicus.Korea began to raise ca비e about 1,800-2,000 years ago and those cattle were supposed to be brought to Korea by nomadic people from China.The first export of Korean native cattle was made to Japan during the period of the Koryo Dynasty (A.D. 918-1,392).From the gene frequency data of blood groups and serum protein polymorphism of East Asian cattle, it has been demonstrated diat the Korean native cattle have greatly contributed to the gene pool of the Japanese native cattle, especially the Japanese brown cattle (Abe et al., 1968;Namikawa, 1972).The Korean native cattle now play an in^)ortant role as a beef producer and as a genetic resource in Korea.
The olgective of this study was to evaluate milk protein genetic polymoiphisms as genetic marker in the Korean native cattle, and to con甲 are the milk protein gene frequencies of the Korean native cattle with those of in中orted ca버e breeds raised in Korea and Japanese brown ca비e.In addition, this paper deals with die finding of a new ^-casein variant

Milk samples
Individual milk san甲les from a total of 280 cows were randomly collected from Alpine Experiment Station and commercial forms of Kang Won Do district.Skim milk was separated from the fat by centrifugation of whole milk at 4°c.The skim milk was stored frozen at -20X3 until electrophoresis.

Genetic marker typing
Milk proteins examined in this study were as follows; QS]-CN, g・CN, x-CN and g-LG.Simultaneous phenotyping of as(-CN,缶CN, x-CN and g~LG genetic variants was performed by slightly modified technique of thin layer of starch urea gel electrophoresis originally described by Aschaffenburg and Thymann (1965) and Aschaffenburg and Michalak (1968).Resolution of gCN A variant into A1, A2 and A3 was achieved by starch gel electrophoresis using acid buffer system described by Arave (1967) with a slight modification.

Statistical analysis
Gene frequencies at the four milk protein loci were determined by single gene counting method (Pirchner, 1983).The genotype distribution within codominant systems was examined for the Hardy-Weinberg equilibrium by chi-square tests (Falconer, 1989).Comparison of gene frequencies for the four milk protein loci between breeds was acconplished using two-way contingency tables (Steel and Toirie, 1980).

Results and Discussion
Starch gel electrophoretic method was used to separate the genetic variants of as广CN,，CN, x-CN and 夕-LG in Korean native cattle.The electrophoretic patterns of as广 CN, g-CN,此-CN and g・LG genetic variants separated on alkaline urea gel are presented in figure 1 and 2. The good separation of the g-LG bands from the leading 0-CN band is shown in figure 2. Figure 3 and 4 show the electrophoretic patterns to resolve the g-CN A genetic variants on acid urea gels.(Thompson et aL, 1962) and Red Danish cattle (Farrell et al., 1971).The as^CN D variant was discovered in Flamande cattle, and shown to occur in low finequency (Grosclaude et al., 1966).The rare aS]-CN E variant occurred in the milk of Yak and Bali ca비e (Grosclaude et al., 1976;Bell et al., 1981).The predominant gene of as「CN in Korean native cattle was asrCN B with a frequency of .846,whereas as,-CN C was in low frequency (.154).The as广CN B variant is predominant in Bos taurus, but asrCN C is in Bos indicus (Aschaffenburg, 1968).In the genotypic frequencies, homozygous BB accounted for 72.5% of the population followed by heterozygous BC with 24.3% and homozygous CC with 3.2%.Sevoi genetic variants of ff-CN are known so far.Ihese variants differ by one or more charged amino acids (Eigel et al., 1984), which makes it possible to separate the ff-CN variant The known genetic variants of，CN are more numerous than those of the other caseins and their differentiation by gel electrophoresis were conq)licated.In alkaline gels, they migrate in the order A1 = A2 = A3 > B > D, E > C.However, in acid gels (Kiddy, 1975), their order is C > B = D > A1 = E > A2 > A3.Thus, although A variants can be differentiated from B, C, D and E by electrophoresis under alkaline conditions, acidic conditions are required for differentiation of A variants.Among the seven known genetic variants of 夕-CN, ttiree genetic variants, A1, A2 and B, were observed, h addition, as shown in figure 3 and 4, a new variant of g-CN was found in Korean native cattle.This new 夕-CN band had a much slower electrophoretic mobility than the 夕-CN A3 band in acid gel electrophoresis.Hence the allele controlling it was designated 夕-CN A4.This band had the same colour as 夕・CN when stained with Amido Black 10 B in the same way as did ff-CN A1, A2, A3 and B. The new 夕-CN A4 variant does not seem to be the same as ff-CN C, D and E according to conq)arisons of measured distances of the migration.Aschaffenburg (1968) proposed a multiple allele theory of inheritance with the types controlled by 夕-CN A1, A2, A3, B and C. Enough family data were not available to study the inheritance of 0-CN A4.However, according to the phenotypic exhibition of 3 hetero2ygote types, ^-CN A1 A4, A2 A4 and BA4, the occurrence of an additional 夕-CN allele is assumed.And its firequency within the population was estimated to be .048.Milk protein gene can be subject to a deletion, a duplication or an insertion.In some cases this leads to a change in the amino acid composition of the protein, resulting in a new genetic variant Ihere is a variation in the histidine contait of ff-CN A, found by sequencing the variants of 夕-CN.Using ^-CN A2 as the base sequence, 8-CN A* has a histidine replacing proline at position 67, and fi-CN A3 has a glutamine replacing histidine at position 106 (Eigel et al., 1984).Thus it will be interesting to cod耳)are the amino acid conq)osition and the primary structure of fi-CN A4 variant with those of &CN A1, A2 and A3 variants.The informations on the genetic control and physicochemical background for very slow mobility of the new variant are not yet available.Therefore, fiirflier work on the genetic and biochemical mechanisms of this new g-CN A4 variant is in progress.On the other hand, a new ^-CN A4 variant seems to be the same as 夕・CN A1 reported by Abe et al. (1975) in J枣 anese brown cattle, according to the conq)arisons of measured distances of the migration on photograph.In the g-CN locus, the A2 allele is predominant (.666) and the B allele 枣pears at a relatively low frequency (.070).Both Aschaffenburg (1968) and Li and Gaunt (1972)  five major Western dairy breeds.In both studies.ff-CN A1, A2 and B accounted for over 95% of the g-CN,)9-CN A2 occurs most often, followed by variants A1, B, C and A3.The 8・CN D variant was discovered in the milk of a few Deshi and Boran cows (Aschaffenburg, 1968) and the 8・CN E in Italian Piedmont cattle (Voglino, 1972).Among the nine different genotypes of 8・CN locus, the most predominant genotypes were homozygous A2A2 and heterozygous A*A2 with frequencies 41.1 and 33.6.The majority of the A2 allele for 8-CN was found in combination with the A1 allele.For g-CN B variant, there were .3%homozygous for B and heterozygotes of combinations of A1, A2 and A4 accounted for 13.2% of the 8・CN.The new 8~CN A4 allele appeared only as heterozygous A*A4, A2A4, BA4 with frequencies 1.8, 6.8 and 1.1%, respectively.In the *-CN locus, two x-CN alleles, A and B, were found in Korean native cattle.However, the *-CN C and E alleles were reported by Putz et al. (1991) in Simmental cattle.The *-CN A allele was observed with a frequency of .648,but *-CN B allele showed to occur in low frequency (.352).The *-CN A allele tends to be predominant in most breeds except in the Jersey, Normande and some South African zebu cattle (Aschafifenburg, 1968).For genotypic frequencies, there were 46.8% for heterozygous AB, 41.4% for homozygous AA and 11.8% for homozygous BB.Among the nine alleles for &LG, two alleles, &LG A and B, were observed in Korean native cattle, whereas the C, D, E, F, G, H and W alleles of 8・LG were not identified.The predominant 8-LG allele was g・LG B with .852frequency, while allele frequency of g・LG A was very low (,148).The predominant genotype of g・LG locus was homozygous BB with 72.5% frequency.There were 25.4 % heterozygous AB but only 2.1% homozygous AA for 8-LG.As a consequence, in Korean native cattle population the most prevalent alleles were B for asrCN, A for 8-CN (A2 for the subset), A for *-CN and B for ff-LG.The observed and expected genotype frequencies showed no significant differences (table 2).According to the Hardy-Weinberg law, the Korean native cattle populations were in genetic equilibrium.Gene frequencies for genetic variants of the four milk protein loci were con甲ared with those rq)orted in the literature for inq)orted cattle breeds raised in Korea and Japanese brown cattle (table 3 and 4).The Korean native ca비。population showed significant differences in the different milk protein gene frequencies con甲ared with those of Holstein breed.Significant differences in gene frequencies between the Korean native ca비。and the three inq)orted beef cattle breeds were also observed at milk protein loci, with the exception of asrCN locus in Charolais and 8-LG locus in Augus.Therefore, the genetic constitution of the Korean native cattle was considered to deviate remaifcably ftom those of inq)orted cattle breeds raised in Korea.However, gene frequencies in Korean native cattle were very similar to those rq)orted in the literature for the Japanese brown cattle.There were no significant differences in gene frequencies for milk protein loci between two breeds, except for 8~CN locus.The &CN B allele was found in Korean native cattle, whereas the 8-CN C allele in Japanese brown cattle.The 8-CN A' allele had the higher frequency in Korean native ca비e, while the g-CN A2 allele in Japanese brown cattle.Interestingly, the new g・CN A4 gene wdiich is supposed to be one of the characteristics of Korean native cattle, exhibits an extremely low frequency (.011) in the Japanese brown cattle.This supports the idea that there has been a genetic connection between the two breeds.The occurrence of this new j9-CN A4 allele in the Japanese brown cattle can be explained that Korean native cattle were used in the development of the Japanese brown cattle.From the data using the frequencies of blood groups and serum protein polymorphisms, Abe et al. (1968) reported that the Korean native cattle have contributed to the gene pool of the Japanese brown cattle.Also, Ito et al. (1988) reported that the Japanese brown cattle had a closer genetic relationship with the Korean native cattle than with Simmental using the frequencies of 17 blood groups and blood protein loci.Results from this study provide a good evidence for supporting the previous studies.Consequently, this study and others indicate that the genetic constitution of the Korean native cattle were Japanese brown cattle originated from Korean native very similar to those of the Japanese brown cattle, and cattle.

TABLE 1 ,
GENE FREQUENCIES AND OBSERVED NUMBER OF THE as,-, *-CN AND ff-LG LOCI IN KOREAN NATIVE CATTLE

TABLE 2
. GENOTYPE DISTRIBUTION AND HARDY-WEINBERG EQUILIBRIUM TEST FOR THE FOUR MILK PRO■「티N LOCI IN KOREAN NATIVE CATTLE Gene frequencies for genetic variants of the four milk protein loci are summarized in table 1, and genotypic frequencies are shown in table 2. In this study, o끼y two asrCN variants, B and C, were found in milk san耳)les of Korean native cattle.The asrCN A variant occurs in very low frequency in both Holstein

TABLE 3 .
GENE FREQUENCIES FOR THE FOUR MILK PROT티N LOCI IN SIX DIFFERENT CATFLE BREEDS

TABLE 4 .
COMPARISONS OF GENE FREQUENCIES BETWEEN KOREAN NATIVE CATTLE AND FOR티GN CATTLE BREEDS FOR THE FO니R MILK PROTEIN LOCI