Genetic Relationships of Cattle Breeds Assessed by PCR-RFLP of the Bovine Mitochondrial DNA D-loop Region

To investigate the genetic relationships among various cattle breeds, bovine mtDNA D-loop region was used in 411 animals of 18 cattle breeds, including 8 Asian Bos taurus, 7 European Bos taurus, 1 Asian Bos indicus, and 2 African Bos indicus. The size of amplified PCR products from mtDNA D-loop region was 964 bp and the products were digested by 15 different restriction enzymes. Two different band patterns were identified in eight restriction enzymes (BstXI, Hae III, Msp I, Apa I, Taq I, Alu I, BamH I, EcoN I) and the rest of restriction enzymes showed more than 3 different band patterns among which Apo I and MspR9 resulted in 7 different restriction patterns. The genotypes, number of haplotype, effective number of haplotype, and degree of heterozygosity were analyzed. Based on all the PCR-RFLP data, different haplotypes were constructed and analyzed for calculating genetic distances between these breeds using Nei's unbiased method and constructing a phylogenetic tree. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No.


INTRODUCTION
With the fast growth of molecular techniques, evolutionary and phylogenetic studies have been growing dramatically, mainly using the mitochondrial DNA (mtDNA) variations.The mtDNA is maternally inherited and its substitution rate is higher than that of nuclear DNA (Brown et al., 1979;Giles et al., 1980).Also, there are few important proteins that are synthesized from the mtDNA (Hutchison et al., 1974).Because of these characteristics, mtDNA variation was used to identify the genetic relationships between and within species of mammals including human.Also, there is absence of crossovers between mtDNA and therefore the mtDNA is the most efficient material to investigate the genetic diversity of the related groups (Brown et al., 1979) Anderson et al. (1981) initially determined the human mtDNA sequence of 16,569 bp and found 13 protein coding regions including 12S, 16S rRNAs, 22 tRNAs, Cytochrome c oxidase subunits I, II, and III, ATPase subunit 6, and Cytochrome b.Also they found bovine mtDNA was consisted of 16,338 bp and there was similarity of protein coding regions between human and bovine but low similarity in D-loop region between them (Anderson et al., 1982).Bob et al. (1982) also found the total length of mouse mtDNA to be 16,295 bp, which is similar in size with human mtDNA but the translational start codon was AUN, which means any of A, G, C, and T could be in the third base of the codon.
Regarding domestication of cattle, Kikkawa et al. (1995) insisted the multiple domestication processesoccurred but the theory lacks any supporting evidence.Loftus et al. (1994a, b) also gave a controversial opinion for the domestication of cattle breeds.They reported that the divergence between the Zebu and Taurine breeds was traced back to 200,000 years ago, or possibly a million years ago.Through mtDNA D-loop analysis, Indian Bos indicus was totally different from African Bos taurus (Bradley et al., 1996) indicating two different ancestors.Based on the mtDNA D-loop variation, Troy et al. (2001) indicated that European cattle breeds were totally different from African breeds and showed more similarity with middle east breeds.
Although Kikkawa et al. (1995) and Mannen et al. (1998b) reported phylogenetic relationships of Japanesecattle with other breeds, not much have been published for the origin of Asian cattle.However, Mannen et al. (1998b) reported that the Japanese black cattle are less related with European cattle breeds, indicating that Japanese black cattle are very important population for investigation of the origin of East Asian cattle.

Genetic Relationships of Cattle Breeds Assessed by PCR-RFLP of the Bovine Mitochondrial DNA D-loop Region
due to lack of experimental sample size.Therefore, the main purpose of our study was to investigate the haplotype frequencies using PCR-RFLP polymorphisms of mtDNA Dloop region and to estimate the origin of Hanwoo by measuring the genetic distances in comparison with other cattle breeds.
The Polymerase Chain Reaction was conducted in 50 µl volumes, each containing 100 ng of genomic DNA, 10× PCR buffer (100 mM Tris pH 8.9, 50 mM KCl, 15 mM MgCl, 0.01% gelatin, 0.1% Triton X-100, 10 mg/ml BSA), 10 pmole of each primer, 200 µM of dNTPs and 1 unit Taq DNA polymerase (Promega, USA).The cyclic condition of PCR includes a first denaturation step of 5 min at 94°C followed by 30 cycles, each consisting of 1 min at 94°C, 2 min at 56°C, and 90 second at 72°C and then, a final extension step of 10 min at 72°C using PTC 200 peltier thermal cycler (MJ Research, USA).

Restriction endonuclease digestion and gel electrophoresis
Fifteen restriction endonucleases (Hsp92 II, BstX I, Hae III, Mbo I, Msp I, Sau96 I, Apa I, Taq I, Alu I, Dde I, Hinf I, BamH I, Apo I, MspR9 I, EcoN I) were used for this study.After the PCR experiments, each products were completely digested in a total volume of 15 µl including 3 µl of the PCR products, 2 units of each endonucleases and the appropriate buffers for 3-6 h at the recommended digestion temperature.The resultants of digestion were electrophoresed in a 3% Metaphore agarose gel or 5% Polyacrylamide gel, and then visualized with ethidium bromide for the genotyping of each sample.

Phylogenetic analysis
Using the records of genotypes of individuals from various breeds, POPGENE package (Yeh FC et al., 1997) was used to analyze the haplotype frequency at different loci for the various breeds and genetic diversity was determined.The genetic distance among the breeds was calculated by the method of Nei (1978).Based on the calculated genetic distance, phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al., 2001).

Variation of mtDNA D-loop region
Based on the PCR-RFLP analysis of mtDNA D-loop region, observed number of haplotypes (Na), effective number of haplotypes (Ne), heterozygosities (H) were calculated (Table 2).The percentages of polymorphic loci, which show polymorphism within breed, were also calculated.For the calculated Ne, Limousin has the lowest value as 1.152 and Luxi has the highest value as 1.713.The H value and percentages of polymorphic loci were relatively high in Asian breeds as compared to European and Bos indicus breeds.These high H value and percentages of polymorphic loci were explained by Watanabe et al. (1985).They indicate that Japanese black cattle and Japanese Shorthorn breeds have higher within breed variations because they are crossed with European breeds such as Simmental, Ayrshire, Devon, Brown Swiss, and Shorthorn.These results also supported by the mtDNA D-loop sequence analysis among breeds (Jung et al., 2002).They explained that the genetic variations in Hanwoo and Japanese black cattle were higher than other European breeds and the reason for the high mtDNA variation in Hanwoo is the introgression of other foreign breeds during past breeding processes.Therefore we assume that Asian breeds have more genetic variation in maternal lines.This also indicates the diverse genetic background in Asian breeds than European and Bos indicus breeds.Within the Asian breeds, Hanwoo and Yanbian cattle shows high percentages of polymorphic loci, indicating that these two breeds have diverse genetic background in maternal lines.
Table 3 shows the genetic variations in each locus for the 18 breeds used in this study.Results shows the lowest Ne value, 1.005, was observed in TaqI locus and the highest value, 2.116, was observed in MspR9I locus.Similar trends were observed in Ht values.Gst value, indicating the level of genetic divergency, was calculated.Form the Gst value, indirect gene flow (Nm) in each generation was presented.The result indicates the locus that has small number of haplotypes were largely affect for the gene flow.

Genetic relationships among breeds based on the D-loop polymorphism
Based on the RFLP haplotype results, genetic distances among cattle breeds were calculated using Nei's unbiased measurement of POPGENE software (Table 4).Results indicate that the Korean cattle (Hanwoo: HW) is closely related with Korean Black cattle (KBC), Yanbien cattle  YAN 0.0213 0.0231 0.0553 0.1031 LUXI 0.1180 0.1637 0.2216 0.3377 0.1536 NAN 0.2742 0.2738 0.3553 0.5053 0.2370 0.0964 CBK 0.0150 0.0563 0.0847 0.2204 0.0762 0.1027 0.3085 AG 0.0826 0.0677 0.1367 0.0685 0.0347 0.2722 0.3743 0.1686 BS 0.0682 0.0428 0.0607 0.1311 0.0427 0.1922 0.3150 0.0960 0.0955 CHA 0.0672 0.0599 0.1193 0.0716 0.0349 0.2359 0.3965 0.1299 0.0075 0.0661 SIM (YAN), synthetic breed among Charolais, Brahman, Hanwoo (CBK) with the genetic distance values of 0.0203, 0.0213, and 0.0150, respectively.Also closely related genetic distance, 0.0499, was observed between Japanese Black Wagyu (WAG) and Korean black cattle (KBC).Interestingly, CBK breed was very closely related with Hanwoo with the genetic distance of 0.0150.This is due to the fact that only males from Brahman and Charolais were used in the CBK breed formation.Therefore only Hanwoo mtDNA, inherited as maternal fashion, can be transmitted to the next generation.Previously, Han et al. (1996) (1975) that Japanese Wagyu breeds were more highly influenced by European breeds based on the milk protein polymorphism.Also, similar resultswere presented by Kikkawa et al. (1995) that genetically close breeds were more likely to cluster in the phylogenetic tree.Brown et al. (1979) reported mutation of mtDNA was 2-4% per million years.Take this value for the calculation of divergence times among breeds, divergence between European and Zebu breeds was occurred 300,000-500,000 years ago.This divergence was also estimated 2-3 million years ago based on the fossil records.The difference of two above divergence time was based on the different substitution rate in each nucleotide in mtDNA.Before the domestication of Zebu cattle, the divergence between Zebu cattle and European breeds were occurred and this hypothesis was also supported by β-chain structure of hemoglobin.Loftus et al. (1994a, b) also estimated the divergence time between Asian breeds and Asian Zebu was occurred 0.2-1 million years ago.
The study on mtDNA D-loop revealed higher H value and percentage of polymorphic loci in Asian breeds as compared to European and Bos indicus breeds.Based on the D-loop polymorphism, the close genetic relationship of Korean cattle (Hanwoo) was observed with Korean Black cattle, Yanbien cattle, Charolais,and Brahman.The phylogenetic tree analysis showed that the Japanese brown cattle and Ceju Black cattle may have closer relationship with European breeds than Asian breeds.

Table 1 .
Haplotype frequencies of each restriction enzyme by the multiple-population descriptive statistics with 18 cattle breeds * Na = Observed number of haplotypes. 1 Ne = Effective number of haplotypes. 2 H =Nei's (1973)gene diversity.

Table 3 .
Summary of the statistical analysis on the genetic variation for all loci among 411 individuals of 18 cattle breeds Na = Observed number of haplotypes. 1 Ne = Effective number of haplotyes. 2Nm = estimate of gene flow from Gst. e.g., Nm = 0.5(1-Gst)/Gst. *
indicated that Hanwoo and Yanbian cattle have a common ancestor.In our analysis, the genetic distances from Hanwoo and Yanbian cattle to Bos indicus breeds or European Bos taurus breeds are similar, supporting that Hanwoo and Yanbian cattle breeds evolved from a common ancestor.Phylogenetic trees were constructed using Nei's genetic distance matrix in POPGENE program (Figure 2), recognizing two major clades, namely Bos indicus breeds versus European and Bos taurus breeds, excluding outgroup of Luxi and Nanyang breeds.Japanese Brown Cattle and Ceju Black Cattle (CBK) show more close relationship with European breeds (Angus, Charolais and Holstein breeds) than Asian breeds (Hanwoo, CBK, Japanese Black Wagyu, Yanbien Cattle).This result was supported by Abe et al.