Discrimination of the commercial Korean native chicken population using microsatellite markers
© Choi et al.; licensee BioMed Central. 2015
Received: 23 December 2014
Accepted: 20 January 2015
Published: 5 February 2015
Korean native chicken (KNC) is a well-known breed due to its superior meat taste. This breed, however, owing to a low growth rate, has a high market price. In order to overcome this disadvantage, the National Institute of Animal Science (NIAS) in Korea developed a commercial KNC breed, named Woorimatdag version 2 (WM2), an upgraded version of the Woorimatdag (WM1) breed and the WM2 was created by crossing the KNC with meat type breeds. This study aims to discriminate between WM2 and other chicken breeds using microsatellite (MS) markers.
A total of 302 individuals from eight Korean chicken populations were examined. The genetic diversity and population structure analysis were investigated using Cervus, API-CALC, STRUCTURE, PowerMarker programs.
Based on heterozygosity and polymorphic information content (PIC) values, 30 MS markers were initially selected from 150 markers. The identified average number of alleles (Na), expected heterozygosity, and PIC values for the WM2 samples were 7.17, 0.741, and 0.682, respectively. Additionally, the paternity of individuals was assigned with a success rate of greater than 99% using 12 markers, the best minimum number of markers. The 12 selected markers contained heterozygosity and PIC values above 0.7 and probability of identity values around zero. Using these markers, the determined probability of identity (PI), PI half-sibs , and PI sibs values were 3.23E-33, 5.03E-22, and 8.61E-08, respectively.
WM2 is well differentiated with respect to other chicken breeds based on estimated genetic distances. The results presented here will contribute to the identification of commercial WM2 chicken in the market.
KeywordsDiscrimination Diversity Microsatellite Korean native chicken Woorimatdag
Recently, chicken meat consumption in Korea has rapidly increased to 12 kg per capita due to consumer preferences for healthy white muscle meat . In comparison to red meat, chicken meat is considered a healthier option because of lower fat, cholesterol, and iron levels . Presently, approximately 90% of the Korean poultry industry contains imported chicken breeding stocks. The breeds that existed before the Korean War (1950–1953), unfortunately, are almost all extinct. Since 1992, a Korean native chicken (KNC) conservation project was launched by the National Institute of Animal Science (NIAS) in an attempt to restore local chicken breeds. Recently, five KNC lines and seven others originally imported in the 1960s have been restored . Consumers tend to pay more for the KNCs because of their good taste. The low productivity of the native breeds, however, was disadvantageous for farmers trying to meet feeding and consumption rates. In order to overcome these disadvantages, NIAS developed the Woorimatdag version 1 (WM1) chicken population. WM1 was a commercial, KNC population generated from crossbreeding fast growing native male chickens and good tasting female chickens with increased egg production. WM1 chicken grows faster, reaching the marker weight of 1.8 kg, than the purebred KNC . Moreover, WM1 chickens produce good quality meat with a high oleic acid content, which improves both taste and water holding capacity . Jung et al.  reported that WM1 chickens have a significantly higher content of arachidonic acid and meat flavor than commercial broilers (Br). NIAS recently developed Woorimatdag version 2 (WM2) chickens, a modified version of WM1 chickens with increased growth rates.
Traditional methods to identify chicken breeds focused on general appearances such as feather color, shank color, and body type . Because of the process of distribution and market, chicken breeds cannot be effectively classified based on the appearance of meat. Recent advances in molecular biology techniques, however, have provided new opportunities to assess genetic variability at the DNA level . Therefore, many groups have attempted to discriminate breeds using molecular genetic markers. For example, Korean cattle and pork industries developed discrimination and traceability systems using microsatellite (MS) markers [8,9].
MS markers or simple-sequence repeat (SSR) markers, are highly polymorphic, one to six base pair repeats, widely used since they are numerous, randomly distributed in the genome, and show co-dominant inheritance [10,11]. In addition, MS markers were used in the construction of linkage map of quantitative trait locus (QTL) studies . MS markers may be useful in discriminating individuals. The International Society for Animal Genetics (ISAG) has recommended 30 MS markers for breed identification .
In a previous study, 97 MS marker variations, including the 30 MS ISAG recommended markers were investigated in 12 chicken populations. While the majority of the 12 population studies were purebred, the commercial chickens were different, products of three- and four-way crosses. Therefore, in this study, the commercial KNC population, WM2 was investigated to discriminate it from the other chicken populations in the market.
Sample collection and DNA extraction
A total of 302 individuals from eight Korean chicken population (187 WM2, 17 WM1, 13 Hanhyup-3 (Hh), 14 Hyunin (Hn), 14 Rhode Island Red (RIR), 15 Cornish Black (CoL), 15 Cornish Red (CoR), 17 Ogye (O) and 10 Br) were examined. Chicken populations care facilities and procedures met or exceeded the standards established by the Committee for Accreditation of Laboratory Animal Care at National Institute of Animal Science (NIAS) in Korea. The study also was conducted in accordance with recommendations described in “The Guide for the Care and Use of Laboratory Animals” published by the institutional Animal Care and Use Committee (IACUC) of NIAS (2012-C-037) in Korea. Genomic DNA was extracted from embryo tissues of WM2 using the PrimePrep™ Genomic DNA isolation kit for tissue (GeNetBio, Korea) and blood samples of other population using the PrimePrem™ Genomic DNA isolation kit for blood. The concentration of DNA samples was measured using NanoDrop 2000C spectrophotometer (Thermo Scientific, USA) and stored at −20°C.
Microsatellite (MS) marker genotyping
Primer information for 30 microsatellite markers used in this study*
Forward (5′ → 3′)
Reverse (5′ → 3′)
Allele size (bp)
The Polymerase Chain Reaction (PCR) was performed in total volume of 20 μL, 50 ng of genomic DNA, 10 pmol of fluorescent dye (FAM, VIC, NED, PET) labeled modified forward primer and normal reverse primer (Applied Biosystems, USA), 2.5 mM of each dNTPs (GeNet Bio, Korea), 10 X reaction buffer (GeNet Bio, Korea), 2.5 unit of prime Taq DNA polymerase (GeNet Bio, Korea). The PCR was performed in an initial denaturation at 95°C for 10 min followed by 35 cycles of 30 sec of denaturation at 95°C, 30 sec of annealing at 60°C, 30 sec of extension at 72°C and final extension at 72°C for 10 min using My-Genie 96 Thermal Cycler (Bioneer, Korea). The PCR products were initially electrophoresis on 3% agarose gel with ethidium bromide (EtBr) and confirmed whether they gave single PCR DNA band under the UV light. When the bands were clearly appeared, further genotyping was performed. For the microsatellite genotyping, more than 20 times diluted PCR products were used. The genotyping reaction contained 1 μL of diluted PCR products, 10 μL of Hi-Di™ Formamide (Applied Biosystems, USA) and 0.1 μL of GeneScan™-500 LIZ™ size standard marker (Applied Biosystems, USA). After dilution, genotyping reaction mixture was denatured for 2 min at 95°C and fragment analysis was performed using capillary array in Genetic analyzer 3130xl (Applied Biosystems, USA). The MS genotypes were identified using GeneMapper ver.3.7 (Applied Biosystems, USA).
Genetic diversity and population structure analysis
The genotyping data were used to estimate mean number of allele (Na), Hexp, observed heterozygosity (Hobs) and PIC using Cervus 3.0 program . The expected probability of identity values among genotypes of random individuals (PI), random half sibs (PI half-sibs ) and random sibs (PI sibs ) were calculated using API-CALC (Average Probability of Identity-Calculate) ver 1.0 . Moreover, we used both model-based and non model-based methods to describe the diversity between pre-defined genetic clusters. We used STRUCTURE software v. 2.3  for model-based and DAPC (Discriminant Analysis of Principal Components) program implemented in adegenet R-package [18-20] for non model-based method. STRUCTURE results were also used to assess population structure of Korean breeds.
DAPC analysis was preceded by the execution of the K-means clustering algorithm implemented in adegenet to identify an optimal number of genetic clusters to describe the data. For this purpose, we ran K-means sequentially with increasing number of clusters. Different clustering solutions are compared using Bayesian Information Criterion (BIC). The optimal number of groups matches the lowest BIC value. Also, genetic distance  values were computed by PowerMarker ver 3.25 . We used the number of loci which differ between two individuals as a measure of the genetic distance between individuals. This was computed using the R package ape . A neighbor-joining tree was then constructed based on the resulting distance matrix using the same package. Genetic distance between breeds was computed using Reynolds genetic distance (which is an allele frequency-dependent distance). NeighborNet graph was computed using splitstree software .
Results and discussion
Polymorphisms of microsatellite markers
The heterozygosity (Hobs and Hexp) and polymorphism information content (PIC) values using 30 MS markers in Woorimatdag version 2 (WM2) chicken population*
No. of allele
Bostein et al.  reported that markers with PIC values >0.5 and Hexp values >0.6 have a high polymorphic information content and are sufficient for breed discrimination. Thus, our results confirmed that all markers, with the exception of ADL0304, have high polymorphic information and suitable allele frequencies and polymorphisms, and can be used to discriminate the WM2 population.
Discrimination of WM2 population
Genetic distance among WM2 and other populations
Matrix of genetic distances among nine populations*
Phylogenetic and structure analysis of nine populations
Since our 12 MS marker combination can effectively discriminate WM2, they can be used for breed identification. Moreover, to the best of our knowledge, this is the first study demonstrating the discrimination of the commercial KNC population, and the results presented here may be applied in the commercial market.
Bayesian Information Criterion
Discriminant Analysis of Principal Components
International Society for Animal Genetics
Korean native chicken
Number of allele
National Institute of Animal Science
Polymerase Chain Reaction
Probability of identity
Polymorphic Information content
Quantitative Trait Locus
Rhode Island Red
Woorimatdag version 1
Woorimatdag verson 2
This study was supported by a grant from the Golden Seed Project (No. 2013005042SB730), Korea Institute of Planning & Evaluation for Technology in Food, Agriculture Forestry & Fisheries (IPET), Republic of Korea.
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