Background and Objective: Korea’s National Institute of Animal Science (NIAS) has preserved two types of purebred chicken strains and the Hanhyup Company has maintained several purebred chicken stains. This study aimed to facilitate the analyses of genetic diversity, parentage and population structure through the isolation and characterization of novel multiplex PCR microsatellite (MS) marker sets for Korean chicken breeds. Materials and Methods: Genotyping of all 27 markers was performed for 469 purebred chicken samples from 20 Korean breeds. Of these, 16 highly polymorphic MS markers were selected based on the number of alleles, expected heterozygosity (Hexp) and polymorphic information content (PIC). The selected markers were classified into two sets of 16 markers each. Results: The expected probability of identity values for the 16 MS markers in random individuals (PI), random half-sib (PIhalf-sibs) and random sibs (PIsibs) were estimated at 1.79×1021, 6.76×1016 and 5.70×108 respectively. Conclusion: Our multiplex PCR marker sets were observed to have favorable applications in the conservation, planning of breeding and traceability systems in the Korean chicken breeds. Additionally, the marker sets will be of considerable use in small- and large-scale population genetic structure analyses.
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Korea’s National Institute of Animal Science (NIAS) has preserved two types of purebred chicken strains: purebred Korean native chickens (KNCs), which include five breeds with different feather colors (red-brown, yellow-brown, gray-brown, black and white) and the “imported and adapted chickens”, which includes two Rhode Island Red breeds, two Cornish breeds and two Leghorn breeds. The latter type were imported in the 1960s for industrialization purposes, adapted to the Korean habitat and maintained as purebred strains1,2. The KNC breeds, owing to their dismal commercial performance, were ignored for many years. Consequently, the populations of these native animal breeds have declined markedly3. The private native chicken breeding-stock company (Hanhyup) is responsible for more than 80% of the native chicken distribution in Korea. The company has maintained purebred chicken strains for commercial purposes for the past 60 years. These are known as the Hanhyup H, F, G, V, S, W, Y, A, 3 (HH, HF, HG, HV, HS, HW, HY, HA and HHC, respectively) and Woorimatdag breeds1. At present, sufficient detailed information about these Korean chicken breeds is not available. Evaluation of the genetic diversity and relationship between local breeds is an important factor in the identification of unique and valuable genetic resources. In addition, in recent times native animals have attracted attention due to the recognition of the importance of breed diversity in the face of ongoing climate change and as potential sources of economically important traits1,4,5. Therefore, it is necessary to be able to investigate the genetic diversity of Korean chicken breeds and classify the breeds accordingly. Genetic marker polymorphisms provide a reliable method to assess the biodiversity within and among chicken breeds. Microsatellites are being used in diversity studies because of their codominant, highly polymorphic nature and availability throughout the genome. Thus, microsatellites have been identified as reliable markers in chickens3,6,7. The identification of these specific markers could aid the selection process for the development of native chickens that are more suitable for the chicken industry in Korea. Therefore, in this study, to facilitate the analyses of genetic diversity, parentage and population structure, novel multiplex PCR microsatellite marker sets for Korean chicken breeds were isolated and characterized.
MATERIALS AND METHODS
Sample collection: A total of 469 purebred chicken samples from 20 breeds were acquired in Korea from Hanhyup and the NIAS. Eight commercial breeds of chickens (HH, HF, HG, HV, HS, HW, HY and HA) were collected from Hanhyup. Samples from 12 purebred breeds were obtained from the NIAS as follows: Red KNC (NR), Yellow KNC (NY), Gray KNC (NG), Black KNC (NL), White KNC (NW), Ogye (NO), Leghorn F (NF), Leghon K (NK), Black Cornish (NH), Brown Cornish (NS), Rhode Island Red C (NC) and Rhode Island Red D (ND) (Table 1).
|Table 1:|| |
Description of the 20 chicken breeds used in this study with 27 microsatellite markers
|Table 2:|| |
Description of the 7 chicken breeds used in the case study with multiplex-PCR marker sets
Multiplex PCR was used on 339 commercial chickens (Table 2). Genomic DNA (g DNA) was extracted from blood samples collected from the wing veins. DNA from the blood was extracted according to the standard protocol provided with the PrimePrep Genomic DNA isolation kit for blood (GeNetbio, Korea).
Marker selection and multiplexing: For the analysis of genetic diversity, 27 microsatellite markers from the FAO/ISAG recommended diversity panel were used. (ISAG/FAO Standing Committee, 2004; Table 3). From these results, 16 highly polymorphic MS markers were selected based on the number of alleles, expected heterozygosity (Hexp) and polymorphic information content (PIC). The selected markers were classified into two sets of 16 markers each (SET1 and SET2). Size fractionation of all 16 markers in each group was made possible by capillary electrophoresis using a combination of fluorescent-labeled primers (Table 6). The possibility of multiplexing the size fractionation by capillary electrophoresis was also considered in the selection of suitable markers.
PCR: All 469 DNA samples were amplified using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). The amplifications were carried out using 15 μL reaction mixtures containing genomic DNA (5-20 ng μL1), primer (10 pM) and 1.5 U Thermo Scientific Maxima Hot Start Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA), which were then subjected to 30 cycles of 30 sec at 95°C, 30 sec at 55°C and 30 sec at 72°C. The multiplex PCR program was the same as the program described above.
Genotyping and statistical analysis: Genotyping of the amplified DNA was performed using an automated Genetic Analyzer 3130xl (Applied Biosystems, USA). The results were obtained using GeneMapper V 4.1 (Applied Biosystems, USA). The genotyped data were analyzed using the Cervus V 3.0 program8 and Excel MS toolkit version 3.19 to calculate the allele frequencies at each locus for each population, the Hexp, the observed heterozygosity (Hobs) and the PIC values. The levels of inbreeding-like effects within subpopulations (genetic distance [Fst]), among subpopulations (within inbreeding [Fis]) and within the entire population (total inbreeding [Fit]) were analyzed by F-statistics10. The STRUCTURE program11 was used to analyze the proportion of the membership within the studied breeds. The expected probability of identity values among genotypes of random individuals (PI), random half sibs (PIhalf-sibs) and random sibs (PIsibs) were calculated using API-CALC (ver 1.0)12. The neighbor-joining method13 was used to construct a phylogenetic tree. Principal coordinates analysis (PCoA) was performed on 7 breeds using the GenAlEx 6.4 program. A correspondence analysis, which is a weighted principal component analysis (PCA), was performed using the allele frequency data for the individuals of all 7 breeds (Table 2) and 16 MS markers with GENETIX software14.
This study was approved by the research Ethics Committee of Hankyung National University 2018-2.
RESULTS AND DISCUSSION
Microsatellite polymorphisms, within and between populations: The microsatellite polymorphism, evaluated by the number of alleles (NA) per locus, mean heterozygosity, PIC and F-statistics values for each breed are described in Table 4. A total of 316 alleles were observed at the 27 microsatellite loci distributed in 469 Korean chickens representing the 20 Korean chicken populations. All the microsatellite loci typed were polymorphic. The NA per locus ranged from 5 (ADL0268) to 20 (MCW0127), with a mean of 11.7 alleles. The mean HExp across loci was 0.797, with estimates per locus ranging from 0.688 (MCW0288) to 0.881 (MCW0104). For HObs, the mean for all loci was 0.547, with a range of 0.359 (GCT0016) to 0.677 (MCW0145, MCW0029). In this study, all the loci had high PIC values (PIC>0.5). Estimation of the genotypic diversity of heterozygosity and PIC value data of the MS markers were previously used for the determination of animal breed selection and animal traceability. Accordingly, markers with PIC>0.5 and HExp>0.6 are the most reasonably informative loci for applications in genetics15-17. F-statistics were estimated in a fixation index as genetic differentiation (Fst), the global heterozygote deficit among the 20 chicken breeds (Fit) and the heterozygote deficit within the breed (Fis) among the 27 MS markers (Table 4). Among these markers, estimation of fixation index has been discovered for Fst, Fit and Fis, with values ranging from 0.119 to 0.382, 0.199 to 0.565 and -0.037-0.337, respectively.
Primer information for 27 selected microsatellite markers in the study
The estimated mean values of Fst, Fit and Fis were 0.233, 0.321 and 0.115, respectively. The breed statistics generated by the 27 microsatellite markers in nine chicken breeds are shown in Table 5. The mean number of alleles (MNA) in each breed ranged from 3.59 (NF) to 6.63 (NY). The two most diverse breeds were the HF and NL breeds, which had the highest mean HExp (0.745 and 0.734), HObs (0.705 and 0.675) and PIC (0.575 and 0.686).
Statistical analysis of heterozygosity (HObs and HExp), polymorphic information content (PIC) and F-statistics value using 27 selected microsatellite markers among the 20 chicken breeds
HEXP: Expected heterozygosity, Hobs: Observed heterozygosity, PIC: Polymorphic information content, Fit: Total inbreeding, Fst: Genetic distance, Fis: Within inbreeding
Statistical analysis of the mean number of alleles (MNA), heterozygosity (HObs and HExp), polymorphic information content (PIC) and within inbreeding (Fis) observed across 27 microsatellite loci for each population
MNA: Mean number of alleles, HEXP: Expected heterozygosity, Hobs: Observed heterozygosity, PIC: Polymorphic information content, Fis: Within inbreeding, HH: Hanhyup H, HF: Hanhyup F, HG: Hanhyup G, HV: Hanhyup V, HS: Hanhyup S, HW: Hanhyup W, HY: Hanhyup Y, HA: Hanhyup A, NR: Red Korea native chicken, NY: Yellow Korea native chicken, NG: Gray Korea native chicken, NL: Black Korea native chicken, NW: White Korea native chicken, NO: Ogye, NF: Leghorn F, NK: Leghorn K, NH: Black cornish, NS: Brown cornish, NC: Rhode Island red C, ND: Rhode Island red D
|Table 6:|| |
Characteristics of microsatellite markers flanking each sarcomere gene and their primer sequences and modifications
Expected probability values among genotypes of random individuals (PI), random half-sib (PIhalf-sibs), random sibs (PIsibs) and total expected probability (PE) for the discrimination of chicken breeds using multiplex-PCR marker SET1 and SET2
Construction of multiplex PCR microsatellite marker sets and case study: Genotyping of all 27 markers was performed for 469 Korean chickens. Table 4 shows the characteristics of different alleles for each marker including the repeat number, frequency, mean heterozygosity (MH) and PIC. Among the 27 loci that were polymorphic in the 20 Korean chicken breeds sampled, 16 highly polymorphic MS markers were selected based on the number of alleles, Hexp and PIC. They were segregated into two sets for multiplex-PCR based on fragment size, genetic variation and peak interpretation (Table 6). For these chicken breeds, a satisfactory expected probability of identity among the genotypes of PI, PIhalf-sibs and PIsibs was suggested to require approximately 16 markers. Thus, the expected probability of the identity values from the 16 MS markers in PI, PIhalf-sibs and PIsibs was estimated as 1.79×1021, 6.76×1016 and 5.70×108 respectively. Overall, the total expected probability (PE) of the identity values was 98.89% for the discrimination of Korean chickens (Table 7). Our study identified multiplex PCR marker sets in Korean chicken breeds.
Figure 1 illustrates the population relationships based on the PCoA using the individual multilocus genotypes of the 16 MS markers, with 2 types of multiplex PCR marker sets from the 7 Korean commercial chicken breeds. With the first type, the two components contributed 29.46 and 12.05%, respectively. By the first component, the HF and HH breeds were confirmed to be clearly separated from the other groups and by the second component, the HY breed was confirmed to be clearly separated from the HS breed.
|Fig. 1(a-b):|| |
Principal components analysis of allele frequencies from (a) SET1 and (b) SET2 in 7 populations using GENETIX
H: Hanhyup H, F: Hanhyup F, S: Hanhyup S, W: Hanhyup W, Y: Hanhyup Y, HCC: Hanhyup 3, WM: Woorimatdag
With the second type, the two components contributed 24.52 and 8.93% respectively and the intergroup relationship showed similarity with the analysis using the first type marker set. From the multidimensional scatter (MDS) plot using 600K SNP genotypes, it can be inferred that HF and HH were classified and maintained together until relatively recently, as their clusters overlap and are spread widely1. Upon comparison, the two results in this study are similar.
Our results identified multiplex PCR marker sets in Korean chicken breeds could be applied in future breeding plans and utilized as discrimination markers for these breeds. This finding suggested that, considering the scale of the domestic chicken market, the 16 identified markers are sufficient and the maintenance of the strains of Korean chickens with appropriate discrimination markers is essential for the conservation of these breeds. Our results indicated that these multiplex PCR marker sets will have considerable applications in small- and large-scale population genetic structure analyses. In addition, they can be used to aid the conservation, traceability and future improvement of these native Korean chicken breeds.
This study identified multiplex PCR microsatellite marker sets for chicken breeds that can be beneficial for investigating the genetic diversity of Korean chicken breeds and classifying the breeds. This study will help Korean chicken breeds to be isolated and characterized. Thus, these specific markers could aid the selection process in the development of native chickens that are more suitable for the chicken industry.
This study was supported by Golden Seed Project, funded by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) [PJ012820052019 (213010053WT251)].