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ISSN : 1225-8504(Print)
ISSN : 2287-8165(Online)
Journal of the Korean Society of International Agriculture Vol.27 No.5 pp.682-686

Characterization of Korean Native Cattle Breeds by Microsatellite Markers on Sex Chromosomes

Seung-chang Kim, Chang-Yeon Cho, Seong-Bok Choi, Ji-Woong Lee*, Jae-Hwan Kim†
National Institute of Animal Science, Rural Development Administration, Wanju 54875, Korea
*Devision of Animal Science, Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 61186, Korea
Corresponding author
October 27, 2015 December 24, 2015 December 24, 2015


This study aimed to identify allele variability and determine the frequencies of microsatellite (MS) markers specific to bovine sex chromosomes in Korean native cattle (KNC; Chikso, Hanwoo and Jeju Black). The three KNC breeds were characterized using 4 MS markers (INRA30, TGLA325, UMN0905 and UMN0929), and allelic richness, allele frequency, heterozygosity and polymorphism information content (PIC) per locus were calculated. The mean observed heterozygosity and PIC values for Chikso, Jeju Black and Hanwoo were 0.541 and 0.542, 0.406 and 0.377, and 0.61 and 0.6, respectively. The heterozygosity and the PIC values of Jeju Black breed were smaller than those of Chikso and Hanwoo. Thus, these genetic parameters were used for the characterization of the breeds and for determining their genetic relationships. The findings of this study might be useful for the conservation, management, and utilization of KNC breeds as genetic resources.

성염색체상의 MS 마커를 이용한 한국재래소의 유전적 특징

김 승창, 조 창연, 최 성복, 이 지웅*, 김 재환†
*전남대학교 동물자원학부


    Rural Development Administration

    Since a few exotic cattle breeds with superior ability are preferred worldwide, indigenous local breeds with low productivity are becoming extinct or endangered; 9% of local cattle breeds have already become extinct and 20% are at a risk of extinction (FAO, 2007). Four Korean Native Cattle (KNC) breeds (Hanwoo, Chikso, Heugu, and Jeju Black) were recently documented in the Domestic Animal Diversity Information System (DAD-IS) of the United Nations Food and Agriculture Organization (FAO; The KNC breeds are classified according to their coat color phenotype (Hanwoo, brown; Chikso, brindle; Heugu and Jeju Black, black) and geographical location (Jeju Black, Jeju Island; Heugu, the Korean peninsula, except Jeju Island). Since the 1960s, livestock improvement project was implemented to enhance the genetic characteristics and performance of the Hanwoo breed. However, the policies to unitize Hanwoo cattle coat color and the introduction of exotic breeds has increased the extinction risk of KNC except Hanwoo (MAF, 2004). Recently, the importance of local cattle breeds as valuable genetic resources was recognized, and hence, research has been focused on their conservation and proliferation. The Jeju Black breed was designated as Natural Monument No. 546 in 2013. The Chikso, Heugu and Jeju Black cattle breeds have been preserved via proliferation by using embryo transfer and in vitro fertilization. Investigation of the color coat expression, intramuscular fat synthesis, and phylogenetic characteristics at the molecular level are being actively conducted for these three endangered KNC breeds (Park et al., 2012; Kim et al., 2013; Lee et al., 2013). Mitochondrial DNA and microsatellite (MS) marker polymorphisms are used for the evaluation of genetic diversity within and across breeds (Groeneveld et al., 2010). In particular, MS markers are considered suitable for analyzing the genetic structure, because of their wide genomic distribution, codominant inheritance, neutrality with respect to selection, large number, and high polymorphism level (Maudet et al., 2002; Wiener et al., 2004; Egito et al., 2007; Dadi et al., 2008; Choi et al., 2012; Pham et al., 2013; Vicente et al., 2008). However, genetic information on the KNC breeds except Hanwoo breed is scarce. In a previous study, we identified genetic markers that could be used to distinguish between domestic and exotic breeds Suh et al., 2013). However, those markers were not suitable for determining the genetic differences among domestic breeds.

    This study aimed to explore the genetic diversity of KNC breeds and to establish the relationship among the three KNC breeds (Hanwoo, Chikso, and Jeju Black) that were raised at four local research institutes and eleven private farms in South Korea using MS markers on sex chromosomes.


    Samples and DNA Extraction

    Blood samples of Chikso were collected from three local institutes (Gangwon Province Livestock Research Center, Jeonbuk Livestock Experiment Station and Chungbuk Veterinary Service Center). Blood samples of Jeju Black were collected from Jeju Special Self-Governing Provincial Livestock Institute. The Hanwoo was additionally sampled for blood from 11 farms (1 to 7) in Yeongju City. Blood samples of these three breeds were randomly collected, preempting intentional sampling of family relationships. Genomic DNA from blood samples was extracted using the DNeasy Blood Kit (Qiagen, Germany).

    Polymerase Chain Reaction Amplification and MS Genotyping

    Thirty MS markers were selected from previous reports (Vaiman et al., 1994; Liu et al., 2002; Liu et al., 2003; Perez-Pardal et al., 2010) and were analyzed to estimate various parameters of genetic diversity. The Polymerase chain reaction (PCR) amplification was performed in a 20μL reaction mixture, which contained 1.5 mM MgCl2, 1 U of AmpliTaq Gold (Applied Biosystems, Foster, CA, USA), 3-10 pmol of each forward (labeled with a fluorescent- colored dye) and reverse primer and 10 ng genomic DNA as a template. Details of the primers are presented in Table 1. The PCR amplification comprised an initial denaturation at 95℃ for 15 min, followed by 35 cycles of denaturation at 95℃ for 60 s, annealing at 55℃ to 63℃ (respective optimal annealing temperature) for 45 s, extension of starters at 72℃ for 60 s and a final extension of starters at 72℃ for 30 min, using the GeneAmp PCR System 9700 (Applied Biosystems). Fluorescently labelled DNA products were carried out electrophoresis using an ABI 3130 xl Genetic Analyzer (Applied Biosystems). Allele sizes of each MS were determined using GeneMapper v4.0 (Applied Biosystems). The formulated allele data was used for statistical analyses.

    Statistical Analyses

    The four markers (INRA30, TGLA325, UMN0905 and UMN0929) showing the genetic difference in KNC breeds were subjected to statistical analysis. Cervus ver. 3.0.7 (Kalinowski et al., 2010) was used to estimate allele frequencies, total number of alleles (NA), mean observed (Hobs) and expected (Hexp) heterozygosities and mean polymorphism information content (PIC) per locus and breed. Genetic structure and the degree of admixture of KNC breeds were investigated using the Bayesian clustering procedure of STRUCTURE ver. 2.3 (Pritchard et al., 2000). Fifty independent runs were performed for each K between 1 and 10, with a burnin period of 50,000 iterations followed by 100,000 iterations of the Markov chain Monte Carlo algorithm. To identify the most probable groups (K) that best fit the data, we used the STRUCTURE HARVESTER (Earl and vonHoldt, 2012), which implements the Evanno method (Evanno et al., 2005).


    Thirty one alleles (24 in Chikso, 21 in Jeju Black and 21 in Hanwoo breed) were identified with four MS markers on sex chromosomes used in this study. Each MS locus showed polymorphism in all the three breeds. The number of alleles per locus ranged from 3 (INRA30) to 9 (UMN 0929) for Chikso, from 3 (INRA30) to 7 (TGLA325, UMN 0929) for Jeju Black, and from 3 (INRA30) to 7 (UMN 0929) for Hanwoo populations. The mean alleles per locus were 6, 5.3, and 4 in Chikso, Jeju Black, and Hanwoo, respectively. The number of typical alleles was 4, 3, and 4 in Chikso, Jeju Black, and Hanwoo breed, respectively. In all, 11 alleles were unique (Table 2).

    The genetic diversity parameters of the three breeds are shown in Table 3. The observed heterozygosity ranged from 0.083 at INRA30 to 0.847 at UMN0929 in Chikso breed; 0.128 at INRA30 to 0.750 at UMN0929 in Jeju Black breed; and 0.360 at UMN0905 to 0.760 at TGLA 325 and UMN0929 in Hanwoo breed. The mean observed heterozygosity for Chikso, Jeju Black and Hanwoo was 0.541, 0.406 and 0.61, respectively. Further, the expected heterozygosity ranged from 0.112 at INRA30 to 0.791 at UMN0929 in Chikso breed; 0.123 at INRA30 to 0.662 at UMN0929 in Jeju Black breed; and 0.513 at INRA30 to 0.757 at TGLA325 in Hanwoo breed. The mean expected heterozygosity for Chikso, Jeju Black, and Hanwoo was 0.578, 0.417 and 0.676, respectively.

    The PIC ranged from 0.106 at INRA30 to 0.755 at UMN0929 in the Chikso breed; 0.118 at INRA30 to 0.603 at UMN0929 in the Jeju Black breed; and 0.395 at INRA30 to 0.698 at TGLA325 in the Hanwoo breed. The mean PICs for Chikso, Jeju Black and Hanwoo were 0.542, 0.377, and 0.6, respectively. The heterozygosity level and PIC were the highest in Hanwoo and the lowest in Jeju Black. The Chikso and Hanwoo breeds showed relatively higher genetic diversity than the Jeju Black breed. The PIC is an indicator of the level of informativeness of a marker (Botstein et al., 1980). All the MS markers used in our study except INRA30 showed high polymorphism (PIC > 0.5) in Chikso and Hanwoo breeds. According to the MS marker selection guideline (Barker, 1994), at least 4 alleles should be identified for each candidate marker. Therefore, three MS markers (TGLA325, UMN0905, and UMN0929) except INRA30 were considered useful for the evaluation of genetic diversity in KNC cattle breeds.

    The population structure among the three cattle breeds were determined using STRUCTURE software. The analyses were repeated 30 times by setting the value of K (the most probable number of identifiable groups) from 1 to 7; the optimal K values were determined using the Evanno method. STRUCTURE HARVESTER software (Earl and vonHoldt, 2012) was used to identify the most probable groups (K) that best fit the population structure data. The highest delta K (ΔK) value was 2 (Table 4) suggesting that the three breeds can be classified into two groups. Assuming K = 2, the proportional contribution of the assumed populations to each breed was computed; the results are shown in Table 5. Each breed was very closely related with the other breeds, supporting the fact that these three breeds might have come from the same origin. MS marker alleles identified in this study could be used to differentiate between Chikso and Jeju Black breeds, but not quite successfully between Chikso and Hanwoo breeds. However, a study based on sequence variation and phylogenetic analysis of mtDNA cyt b gene (Kim et al., 2013) has shown genetic divergence between Chikso and Hanwoo. The largest ΔK values generally indicate the optimal K, but substructures are weakly defined when only a small number of animal or breeds are analyzed (Leroy et al., 2009). Further, the level of genetic diversity varies according to population sizes among breeds. Hence, further studies with large sample size involving larger number of groups are required to determine the genetic variation among KNC breeds.

    At present, little information is available regarding the genetic diversity in KNC breeds. To our knowledge, this is the first study performed using MS markers on sex chromosomes to analyze the genetic diversity among three KNC breeds. Our findings might contribute to understanding the genetic diversity and relationships among KNC breeds and might be useful for the conservation, management, and utilization of KNC breeds as genetic resources.

    적 요

    이 연구의 목적은 성염색체 특이적 초위성체마커의 대립 유 전자 다양성과 빈도를 조사하여 한국 재래소 3품종의 (칡소, 한우 그리고 제주흑우) 유전적 근연관계 및 특징을 조사하여 우리 고유 유전자원으로서의 가치를 구명하고자 하였다. 한국 재래소 3품종은 30개의 초위성체 마커에서 선별된 4개의 초 위성체 마커(INRA30, TGLA325, UMN0905 및 UMN0929) 를 이용하여 식별하였다. 대립 유전자의 다양성, 대립 유전자 빈도, 이형접합도 그리고 다형 정보량(PIC)을 산출 하였다. 칡 소, 제주 흑우와 한우에 대한 관찰 이형접합도의 평균은 각각 0.541, 0.406 및 0.61이었고 PIC의 평균은 각각 0.542, 0.377 그리고 0.6이었다. 제주흑우의 경우 칡소나 한우에 비해 낮은 이형접합도와 PIC를 보여준다. 이들 4개의 초위성체 마커를 이용하여 칡소, 제주흑우 그리고 한우의 품종을 구별에 이용 할 수 있다. 이러한 결과들로 볼 때 한국 재래소 3품종은 가 축유전자원으로서 중요한 가치를 지니고 있으며 이들 품종의 보존, 관리 및 활용에 중요한 기초자료로 이용될 것으로 사료 된다.


    This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01029302)” Rural Development Administration, Republic of Korea and was supported by 2015 Postdoctoral Fellowship Program of National Institute of Animal Science, Rural Development Administration, Republic of Korea.



    Information of five sex-chromosome microsatellite markers.

    Identified alleles in 3 Korean Native Cattle breeds.

    Bold = alleles found only in one breed

    Genetic diversity parameters of identified alleles in 3 Korean Native Cattle breeds.

    NA = number of Allele; NS = number of sample; Hobs = observed heterozygosity; Hexp = expected heterozygosity; PIC = Polymorphism Information Content.

    The estimated Delta K values by Evanno method (2005)

    Delta K = Mean(|LnN"(K)|) / SD(Ln(K))

    Proportional contribution of the inferred clusters (K=2) to the breed studied using STRUCTURE analysis.


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