Discrimination of Korean Native Chicken Populations Using SNPs from mtDNA and MHC Polymorphisms

Korean native chickens are a very valuable chicken population in Korea and their prices are higher than that of commercial broilers. In order to discriminate two commercial Korean native chicken populations (CCP1 and CCP2), single nucleotide polymorphisms (SNPs) from mitochondrial (mt) DNA D-loop sequences and LEI0258 marker polymorphisms in the major histocompatibility complex (MHC) region were investigated. A total of 718 birds from nine populations were sampled and 432 mtDNA sequences were obtained. Of these, two commercial Korean native chicken populations (363 birds) were used for investigation of their genetic relationship and breed differentiation. The sequence data classified the chickens into 20 clades, with the largest number of birds represented in clade 1. Analysis of the clade distribution indicated the genetic diversity and relation among the populations. Based on the mtDNA sequence analysis, three selected SNPs from mtDNA polymorphisms were used for the breed identification. The combination of identification probability (Pi) between CCP1 and CCP2 using SNPs from mtDNA and LEI0258 marker polymorphisms was 86.9% and 86.1%, respectively, indicating the utility of these markers for breed identification. The results will be applicable in designing breeding and conservation strategies for the Korean native chicken populations and also used for the development of breed identification markers. (


INTRODUCTION
With the burgeoning global population, techniques for the identification of highly productive food sources are needed.Many native livestock breeds, which have less productivity and are mostly found in developing countries, have diminished in number or become extinct.Recently, the United Nations called for the identification of all the native livestock breeds in the world in order to develop conservation strategies (http://dad.fao.org).
In Korea, there are increasing demands from consumers for Korean native chickens.For this reason, two commercial Korean native chicken populations (CCP1 and CCP2) were recently developed at the National Institute of Animal Science (NIAS) and from a commercial company in Korea, respectively, by crossing Korean native chickens with meat-type chicken breeds.Their meat prices are 2-3 times more expensive, compared with commercial broilers.However, the recent marketplace availability of CCP1 and CCP2 has proved confusing to consumers.
To discriminate CCP1 and CCP2 from their meat-type breed counterparts at the molecular level, variations in their mitochondrial genome were initially investigated.The mitochondrial genome is maternally inherited and the sequences of mitochondrial DNA (mtDNA) have been extensively used in biodiversity studies of vertebrates (Baker and Marshall, 1997;Mindel et al., 1997;Moore and Defilippis, 1997;Wayne et al., 2002) including chickens and domestic animals (Komiyama et al., 2003(Komiyama et al., , 2004;;Liu et al., 2006;Odahara et al., 2006;Sasazaki et al., 2006;Lei et al., 2007;Wang et al., 2007;Li et al., 2008).Typically, coding genes of the mitochondrial genome are used for phylogenetic studies to distinguish species (Moore and Defilippis, 1997), whereas the control region is considered more suitable for inter-specific population studies (Baker and Marshall, 1997).Advantages of the mtDNA control region as molecular markers include the maternal inheritance of the mtDNA, rapid nucleotide substitutions compared with the nuclear genome, and the absence of recombination (Aquadro and Greenberg, 1983;Lansman et al., 1983;Cann et al., 1984).
In the nuclear genome, the major histocompatibility complex (MHC) has important biological functions associated with immunity, design of effective vaccines, reproductive success and production traits of domestic animals (Bernatchez and Landry, 2003;Piertney and Oliver, 2006).Parasites and sexual selection have been documented to sustain the unusually high levels of MHC polymorphism (Piertney and Oliver, 2006).Particularly, the microsatellite marker LEI0258 of domestic chickens is widely-used for the discrimination of MHC alleles.There are large numbers of alleles identified in this marker, which is located in the MHC region (McConnell et al., 1999).Also, LEI0258 marker amplicons have repeat number variations, indels (insertion or deletion) and single nucleotide polymorphisms (SNPs), which make easy identification of each allele using direct polymerase chain reaction (PCR) and sequencing (Fulton et al., 2006).
In this study, SNPs in mtDNA and LEI0258 polymorphisms of Korean native chicken populations were investigated to develop molecular markers for breed identification.

Experimental animals
A total of 718 chicken samples from nine populations were collected from NIAS, and commercial chicken farms.

DNA extraction and PCR amplification
Genomic DNAs were extracted from blood and liver samples using PrimePrep TM Genomic DNA Isolation Kit (GeNet Bio, Korea) according to the manufacturer's instructions.The primers forward: 5'-AGGACTACGGCTT GAAAAGC -3' and reverse: 5'-ATGTGCCTGACCGAGG AACCAG -3') were used to PCR amplify a 591 bp fragment of the D-loop hypervariable region in mtDNA.Also a new primer set (forward: 5'-TCAGTGATGTCATC GGGAAA -3' and reverse: 5'-TTTTCAGATCGCGTTCC TCT-3') was designed to amplify the LEI0258 marker to investigate the MHC alleles.Both PCR reactions included approximately 100 ng of genomic DNA, 2.5 μl of 10× buffer [Tris-HCl (pH 9.0), PCR enhancers, (NH 4 ) 2 SO 4 , 20 mM MgCl 2 ], 2.0 μl of 10 mM dNTPs mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP), 1 μl of 10 pmol of each primer and 1 U HS Prime Taq (GeNet Bio, Korea) in a 25 μl reaction volume.PCR was performed in a My-Genie96 Thermal Block (Bioneer) with an initial denaturation step at 94°C for 10 min followed by 35 cycles of 30 s at 94°C, 30 s at 61°C, 40 s at 72°C and a final extension step at 72°C for 10 min.The PCR products for mtDNA and LEI0258 markers were electrophoresed on 1.5% and 4% agarose gels stained with ethidium bromide, respectively, and DNA fragments were visualized under ultraviolet light.

DNA purification and sequencing
Purification of PCR products was performed using an Accuprep ® PCR purification kit (Bioneer) according to the manufacturer's instructions.Purified PCR products were also confirmed by using agarose gels for sequencing.In case of MHC alleles, homozygous alleles were considered for sequencing.All the purified PCR products were sequenced by Genotech (www.genotech.co.kr).

Data analysis
The chicken mtDNA D-loop nucleotide sequences and MHC alleles were aligned using the ClustalW program (Thompson et al., 1994) and saved as bioedit format.Nucleotide replacement export data from mtDNA were carried out in haplotype sequences and identical sequences for MHC alleles were considered as the same haplotypes by using MEGA software version 4.0.2(Kumar et al., 2008).To calculate the identification probabilities between CCP1 and CCP2, allele frequencies of these two populations were investigated by PCR-restriction fragment length polymorphism (PCR-RFLP).The RFLP reaction consisted of 10× buffer, 3-5 units of each enzyme and 15 μl PCR product in a total volume of 20 μl.Three selected SNPs at position 225, 239 and 243 were identified by digestion of the mtDNA PCR products with HphI, HpyCH4III and AluI restriction enzymes, respectively.The obtained RFLP fragments were separated on 4% agarose gels stained with ethidium bromide (Figure 1).Also, haplotype frequencies were investigated using the algorithm from Haploview program developed by the Broad Institute (Haploview, USA).

Analysis of mtDNA SNP
Based on the analysis of 432 chicken mtDNA D-loop sequences, a total of 32 nucleotide substitutions were identified and classified in 20 clades (Table 1).Indel (insertion or deletion) mutations were not detected.The 1 Numbers indicate nucleotide base position in mitochondrial D-loop region and hypen (-) represents the identical nucleotide with the type 1 sequence.identified average percentage of polymorphic sites was 5.86% for the 546 bp mtDNA sequences.In Chinese native chicken breeds, the average percentages of polymorphic sites were previously determined as 6.4% and 4.45%, respectively (Fu et al., 2001;Liu et al., 2004).In the D-loop region, three polymorphic positions, 225, 239 and 243, were considered for breed discrimination markers because of the large differences in allele frequencies between the CCP1 and CCP2.Of these three SNPs, five haplotypes were obtained from two commercial populations (Table 2).Four haplotypes (TAT, CGC, TGT and CAT) were found in CCP1 and the highest frequency (71.9%) was the TAT haplotype.In CCP2, TGT and CAT haplotypes were not identified while TAT, CGC and CGT haplotypes were identified.The CGC haplotype had the highest frequency (81.4%) among the three haplotypes.

MHC allele investigation
Polymorphisms and repetitive sequences were identified for the LEI0258 alleles in CCP1 and CCP2 populations by sequencing for homozygous animals.The alleles having Genbank accession numbers were related with serologically identified MHC-B haplotypes (Table 4).The identified   MHC alleles in CCP1 and CCP2 had 13 and 10 alleles, respectively (Figure 2).To calculate breed identification between CCP1 and CCP2, the sum (64.4%) of three alleles (330, 420 and 434) having high allele frequencies in CCP1 and the sum (62.2%) of three alleles (482, 545 and 568) having high allele frequencies in CCP2 were considered.

Calculation of discrimination probability
Three mtDNA markers (SNP225, SNP239 and SNP243) and the LEI0258 marker were evaluated for discriminating  CCP1 and CCP2 populations.These probabilities were calculated based on the estimated allele frequency for each marker (Table 5).The probability of identification within the breeds was estimated by considering minor allele frequencies.Based on the combination of three mtDNA markers in CCP1 and CCP2, the obtained probability of identifications were 68.5% and 64.2% respectively.When the MHC allele was included in the analysis, the probability of identification increased to 86.9% and 86.1% for CCP1 and CCP2, respectively.Previous results indicated that more than 0.8 of identification probability (Pi) were considered as effective for breed identification in cattle (Alves et al., 2002;Sasazaki et al., 2004Sasazaki et al., , 2006)).Presently, the combination of four markers effectively discriminated these two commercial Korean native chicken populations.
In this study, we investigated the probabilities of breed identification using molecular markers derived from mtDNA and MHC alleles for the discrimination of chicken populations.To maintain the valuable native chicken population, molecular markers for breed discrimination would be very useful for an appropriate conservation breeding program as well as for the establishment of molecular markers for chicken breeds in the marketplace.However, more investigation of molecular studies is required to further improve the breed discrimination.

Table 1 .
Mitochondrial D-loop sequence polymorphisms identified in the chicken populations used in this study Clades Nucleotide position in mtDNA D-loop region 1

Figure 2 .
Figure 2. Comparison of allele frequencies for LEI0258 marker between CCP1 and CCP2.Major alleles in CCP1 and CCP2 are indicated by asterisks (*).

Table 3 .
Distribution of clades in each chicken population based on sequencing

Table 2 .
Haplotype frequencies in CCP1 and CCP2 based on RFLP genotyping

Table 4 .
Polymorphisms identified for the LEI0258 alleles in CCP1 and CCP2 populations by sequencing

Table 5 .
Allele frequencies and identification probabilities (Pi) for four effective markers in CCP1 and CCP2