Genomics 114 (2022) 110298

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Genomics
journal homepage: www.elsevier.com/locate/ygeno

Comparative genomics and selection analysis o Yeonsan Ogye black

chicken with whole-genome sequencing
Youngbeom Cho a, b, 1, Jae-Yoon Kim a, b, 1, Namshin Kim a, b, *
a
Genome Editing Research Center, Korea Research Institute o Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic o Korea
b
Department o Bioinormatics, KRIBB School o Bioscience, University o Science and Technology (UST), Daejeon 34141, Republic o Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Yeonsan Ogye (OGYE; Gallus gallus domesticus) is a rare indigenous chicken breed that inhabits the Korean
Chicken Peninsula. This breed has completely black coloring, including plumage, skin, eyes, beak, and internal organs.
Population genetics Despite these unique morphological characteristics, the population o OGYE has declined without in-depth
Selection signature
research into their genome research. Thereore, this study aimed to compare the whole genome o OGYE to
Whole-genome sequencing
Genomic characteristics
12 other chicken populations, including ancestral breed, commercial breeds, Chinese indigenous breeds, and
Yeonsan Ogye Korean native chickens. We ocused on revealing the selection signature o OGYE, which has occurred through
environmental pressures in the Korean Peninsula. Genome-wide selection analysis has identied local adaptation
traits, such as egg development, that contribute to etal viability and innate immune response to prevent viral
and microbes inection in OGYE. In particular, SPP1 (Secreted Phosphoprotein 1), HSP90AA1 (Heat Shock
Protein 90 Alpha Family Class A Member 1), and P2RX4 (Purinergic Receptor P2X 4) could have considerable
involvement in egg development and RNASEL (Ribonuclease L), BRIP1 (BRCA1 Interacting Protein C-terminal
Helicase 1), and TLR4 (Toll-Like Receptor 4) are crucial or the determination o the innate immune response.
This study revealed the unique genetic diversity o OGYE at the genome-wide level. Furthermore, we emphasized
the sustainable management o genetic resources and ormulated breeding strategies or livestock on the Korean
Peninsula.

1. Introduction
The Yeonsan Ogye (OGYE) is a representative Korean native chicken
breed (Gallus gallus domesticus), considered a natural monument by the
Korean government (Protected Species Act No.265), which is also
registered with the Food and Agriculture Organization o the United
Nations (FAO) as Yeonsan Ogye [1]. OGYE can be distinguished rom
other chicken breeds by the complete melanism o beak, eyes, ace,
earlobes, wattle, tongue, shank, toes, eathers, and skin as well as its
bones [2]. In the Joseon Dynasty (1392–1910), OGYE had a royal
connection and was honored. OGYE has conserved a distinct genetic
basis ater hundreds o years o successive adaptation in the Korean
Peninsula [2–4]. OGYE and the Korean native chicken (KNC) were
domesticated in Korea approximately 2000 years ago ater migration
rom their native regions o South-East Asia and China [5]. The two
breeds successully adapted to the environment o the Korean Peninsula,
where; they were mainly raised on small-scale arms. The KNC has
several plumage colors and eather patterns (white, black, yellow-
brown, gray-brown, and red-brown). The KNC is also preserved by the
Korean government as a valuable animal to protect the richness o ge-
netic diversity [5].
Domestic chickens descended primarily rom the wild Red Jungle
Fowl (RJF) o Southeast Asia (approximately 8000 to 5400 BCE) and
have subsequently spread throughout the world [6]. Environmental
pressures and articial selection have generated signicant genome-
wide divergence in these chickens, as those selection pressures
contribute a considerable evolutionary orce to phenotypic and geno-
typic dierentiation [7]. Domestic chickens have developed a substan-
tial number o dierent phenotypic characteristics that infuence
morphology, tness, physiology, egg production, and tolerance to
abiotic and biotic stresses [8].
A previous study reported a selective signature in Tibetan chickens
highlighting several candidate genes in relation to an oxygen-sensing
ability that aids in acclimation to high-altitudes and low-oxygen levels

* Corresponding author at: Genome Editing Research Center, Korea Research Institute o Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic o Korea.
E-mail address: deepreds@kribb.re.kr (N. Kim).
1
These authors contributed equally to this research.

https://doi.org/10.1016/j.ygeno.2022.110298
Received 18 May 2021; Received in revised orm 24 December 2021; Accepted 1 February 2022
Available online 5 February 2022
0888-7543/© 2022 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Y. Cho et al.
[9]. Another study involved indigenous chickens rom tropical countries
and identied candidate genes related to high biological response
sensitivity in the hot and humid environment, such as Brazil, Sri Lanka,
and Egypt [10]. These indigenous chicken studies uncovered breed-
specic genetic material and emphasized the importance o character-
izing the genetic components o indigenous breeds.
Several previous studies have been conducted to uncover the char-
acteristics o OGYE. This breed is recognized as sensitive and easily
intimidated by humankind, and it is susceptible to environmental con-
ditions and rapidly adapting to environmental changes that increase
with its age [11]. Moreover, previous studies on OGYE have shown that
in terms o growth rate variation and egg production, and it was iden-
tied as having a thicker eggshell than those o KNC, Korean White
Leghorn (WLH), and Korean Rhode Island Red (RIR) [11]. The genetic
characteristics o OGYE have been analyzed using whole-genome and
transcriptome map data to understand the genomic dierence and
unctioning o hyperpigmentation in OGYE [12]. Although OGYE has
not been deeply researched as other chicken breeds yet, these studies
have proposed that OGYE conserved unique phenotypic and genotypic
properties as an indigenous breed and has raised the need or urther
research to understand their genomes, which could contribute to theo-
retical population genetics, acilitate local adaptation and economic
traits. This study aimed to: (I) identiy or the rst time the genomic
characterization o OGYE and KNC with whole-genome sequencing data;
and (II) determine key variants/genes underlying the selection signa-
tures o OGYE using the comparative genome-wide analysis.
2. Materials and methods
2.1. Sample preparation and data description
A total o 188 whole-genome chicken samples were used in this
study, o which 120 were newly sequenced and shared by the Animal
Genetic Resources Station, National Institute o Animal Science, Rural
Development Administration (Jeon-Ju, South Korea), according to a
joint research agreement (Approval No: 2012-D-010). The sequencing
data were deposited in the Korean National Agricultural Biotechnology
Inormation Center (https://nabic.rda.go.kr/), and the remaining 68
public whole-genome chicken samples were collected rom Sequence
Read Archive. All sample accession numbers are summarized in Sup-
plementary le1: Table S1. The 120 newly sequenced samples consisted
o nine breeds: 10 Korean native red-brown (KNR), 10 Korean native
black (KNB), 10 Korean native gray-brown (KNG), 10 Korean native
white (KNW), 10 Korean native yellow-brown (KNY), 10 OGYE, 20
Korean Cornish (COR), 20 WLH, and 20 RIR (Supplementary le1: Table
S3). Six o the breeds are representative Korean native chickens, and the
remaining three common commercial breeds are raised in Korea. The
other 68 chickens were included in our breeds, as ollows: 10 RJF, 10
Chinese ghting cocks (CNF), 18 Tibetan chickens (CNT), and 31
Southern Chinese native chickens (CNS) (Supplementary le1: Table
S2). In this study, the extensive comparative analysis included RJF,
ancestral species, and Chinese native chickens based on historical re-
cords stating that these are KNC initially imported rom China [5].
2.2. Data processing and variant calling
We rst used a FastQC [13] to check the quality o the base-paired
sequence. The FASTQ les then were mapped against the Gallus_gal-
lus_5.0 reerence genome (https://www.ncbi.nlm.nih.gov/assembly/
GCF_00000 2315.4/), generating a BAM le through BWA [14]. The
mapped BAM les were assigned to Gallus_gallus_5.0 reerence genome
coordinates using the Genome Analysis Toolkit v4.0.2.0 (GATK4) [15]
package ‘AddOrReplaceReadGroups’ ollowed by removal o potential
PCR duplicates using GATK4 ‘MarkDuplicates’. Then, the ‘FixMa-
teInormation’ was used to veriy the mate-pair inormation between
reads. The ‘BaseRecalibrator’ and ‘ApplyBQSR’ were perormed on the
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Genomics 114 (2022) 110298
BAM les in a two-stage process to minimize systematic bias and to
infuence the assignment o base quality scores made by the sequencer.
The ‘RealignerTarget Creator’ and ‘IndelRealiner’ o GATK v3.8 were
then applied to the BAM les to correct misalignments made by the
genome aligners in the region containing INDELs. Furthermore, the
option ‘HaplotypeCaller’ in GATK4 called all base sites o the reerence
genome and generated gVCF les, including raw SNPs and raw INDELs.
The 188 gVCF les were merged into a single gVCF le using the GATK4
‘GenomicsDBImport’ and then converted to a single VCF le using
‘GenotypeVCFs’. The ‘VariantFilteration’ and ‘SelectVariants’ options
were used to remove alse-positive variants or the ollowing ltration
options: (I) read position rank-sum test (ReadPosRankSum) < -2 (II)
mapping quality rank-sum test (MQRankSum) < -2; (III) phred-scaled
quality score (QUAL) < 30; (IV) quality by depth (QD) < 3.0; (V)
FisherStrand (FS) > 60; (VI) RMS Mapping Quality (MQRankSum) < 40;
(VII) depth o coverage (DP) < 7 && 30 < (DP); and (VIII) genotype
quality (GQ) < 10. VCFtools v0.1.17 was used to perorm variants to
lter by genotyping missing rates o > 20% and minor allele requency
< 1% [16]. We separated SNP and INDEL variants rom the VCF le and
retained only the highest requency alternative SNP allele to maintain
the ollowing imputation. The next step was to impute the missing ge-
notype and phasing haplotype using Beagle v5.1 [17]. Identied SNPs
were annotated on the unctional genomics and protein regions by
SnpE [18]. WGS variant calling was applied to 188 chicken samples
utilizing the GATK Best Practice recommendations and were executed
conservatively to increase statistical power or evaluation o selection
pressure.
2.3. General genomic characteristics
Nucleotide diversity (π) was measured in windows using VCFtools
[16] with a window size o 50 kb, sliding size o 25 kb. The inbreeding
coecient (F) was also calculated using the VCFtools. An individual’s F
value indicates the probability that a pair o genes at a randomly
selected DNA location is identical-by-descent (IBD). PopLDdecay was
used to compute the linkage disequilibrium (LD) pattern using all bi-
allelic SNPs [19], and also to iner the square o the correlation coe-
cient (r2) as suggested. The summary o π, F, and LD are provided or
each population (Supplementary le1: Table S4). For genetic dieren-
tiation among 13 chicken breeds, VCFtools was used to measure the
pairwise xation index value (Fst) [20] with a window size o 50 kb and
a sliding size o 25 kb. We built a phylogenetic tree based on all iden-
tied bi-allelic SNPs using SNPhylo [21] and visualized the phylogenetic
tree with MEGA10 [22]. RJF was the root o the phylogenetic tree, and
we used both the neighbor-joining and maximum-likelihood algorithms
to adopt the phylogenetic tree (Fig. 1A and Supplementary le1: Fig.S7).
2.4. Genomic relationship and population structure
We used astStructure [23] to calculate the admixture analysis based
on a variational Bayesian ramework and estimated rom the number o
genetic clusters (K) K = 2 to K = 7, and each genetic cluster was
calculated using the corresponding cross-validation errors (Fig. 2B).
Plink [24] was used or the principal component analysis (PCA) to
obtain eigenvalues and eigenvectors to conduct the PCA, and the results
were observed using R sotware (Fig. 2A and Supplementary le1: Fig.
S4). PCA is a dimension reduction method by transorming multiple
eatures into a lower-dimensional eature that explains most o the
variance in a large dataset. We used Treemix v1.13 [25] to estimate the
historical relationships among populations, such as gene fow and ge-
netic drit. RJF was used as the root and block size or calculating a
window size o 300 kb (Supplementary le1: Fig.S3). The eective
population size o the chicken populations was calculated separately,
using SMC++ [26] (Fig. 3B). We assumed a constant generation time (g)
o one year and xed per-generation mutation rate o 1.91 × 109 per
generation [27].
Y. Cho et al. Genomics 114 (2022) 110298

Fig. 1. Overview o 13 WGS chicken samples covered in this study, (A) Map shows the geographic distribution o the sampling location across South Asia and East
Asia. (B) Appearance o OGYE with a Black morphological eature (Photo credits by Hitchock Hens). (C) The number o bi-allelic SNPs detected in each chicken
population covering the reerence Gallus_gallus_5.0 or 13 chicken populations.

2.5. Selection signals and gene set enrichment analysis
To identiy genetic loci under positive selection, we perormed the
cross-population composite likelihood ratio test (XP-CLR) [28] and the
cross-population extended haplotype homozygosity test (XP-EHH) [29].
We assumed that the genetic position was equally matched to a physical
position (1 Mb = 1 cm) because o the absence o a chicken genetic map.
XP-CLR v1.0 adopted a 50 kb window with a 25 kb sliding window size
to examine the WGS dataset. We accepted selection regions o the top
0.5% o the empirical distribution o the XP-CLR raw scores and assigned
them to selected candidate gene regions. Genes were ound closest to
selected candidate regions that were considered as candidate genes. We
used the R sotware package rehh v3.2.1 [30] to calculate a raw score o
XP-EHH that targeted a dierential selection signature between two
populations. We accepted the top and bottom 0.5% o the empirical
distribution o XP-EHH, the genetic region, as a selection candidate re-
gion based on the p-value. Genes ound closest to selected candidate
regions were assigned as candidate genes. The selection o candidate
genes detected using these two methods are summarized in (Supple-
mentary le2–le3). To identiy the unctional inormation o candidate
genes, we pooled candidate genes to perorm gene set enrichment
analysis using the Kyoto Encyclopedia o Genes and Genomes (KEGG)
pathway analysis [31]. The KEGG pathway analysis linked the candidate
genes to gene unctions, diseases, and biological pathways and grouped

3
Y. Cho et al. Genomics 114 (2022) 110298

Fig. 2. Genomic stratication and relationship o 13 chicken population, (A) Phylogenetic tree was inerred using the Neighbor-Joining method based on 188
chicken. (B) Genomic structure or 13 chicken breeds assuming the dierent number o ancestral population (rom K = 2 to K = 7). (C) PCA results o all 188 chicken,
(D) all six Korean native chicken breeds.

Fig. 3. LD decay or 12 chicken population and estimate o eective population size. (A) The extent o the LD decay or 12 chicken populations. CNT were excluded
(B) Estimated population size or the OGYE (Red), KNC (Purple), CNS (Blue), and CNF (Orange). (For interpretation o the reerences to colour in this gure legend,
the reader is reerred to the web version o this article.)

them by pathway categories. The statistical signicance level or the

pathway categories was determined using the Bonerroni method with a
p-value o 0.05 (Supplementary le4).

4
Y. Cho et al.
3. Results
3.1. Whole-genome resequencing and identifcation o SNPs and INDELs
The 188 chicken samples were collected rom South Korea, China,
and Indonesia; their raw-resequenced data were analyzed to obtain a
single-nucleotide polymorphism (SNP). In total, 33.65 billion reads o
101 bp (3.39 Tb o sequence) were generated and aligned to the chicken
reerence genome, Gallus_gallus_5.0 (Ensembl release 94). The average
alignment rate was 97.31% and the genome coverage rate was 98.67%
o the reerence genome. Moreover, the average read depth ater
removing duplicated reads was 16.73×. The variant calling process
identied 42,993,594 raw variants, 1,862,730 insertion/deletion poly-
morphisms (INDEL), and 18,902,359 bi-allelic SNPs remained ater
quality control procedures. These 18,902,359 bi-allelic SNPs ltered
with a minor allele requency (MAF) greater than 1% and a missing rate
less than 20%. The ltered variants were included in the urther asso-
ciation analysis (Supplementary le1: Table S1). The number o bi-
allelic SNPs was the highest or the RJF, ollowed by the CNS and CNT
(RJF: 16,195,406; CNS: 15,744,576; and CNT: 14,197,259) (Fig. 1C).
The number o SNPs in RJF was 1.70 times greater than the average
number o SNPs in COR, WLH, and RIR. The commercial chicken breeds
were maintained genetic homogeneity, which is a signature o articial
selection. (COR: 10,901,971; WLH: 9,530,318; and RIR: 8,146,054).
Among the indigenous breeds, the average number o SNPs in OGYE and
KNC had 1.06 times greater than the average number o variants in COR,
WLH and RIR. Also, the number o SNPs in CNS has 1.57 times more than
average number o SNPs in OGYE and KNC (OGYE: 9,564,547; and KNC:
10,539,628). Due to the small population size (only around 2000 OGYE
are saved), OGYE had a similar homogeneous genetic characteristics to
commercial chicken breeds. We used 18,902,359 bi-allelic SNPs with a
high genome-wide resolution, covering all 188 chicken samples or the
ollowing analysis.
3.2. Genomic architecture o chickens
Nucleotide diversity (π) determines the degree o polymorphism
within a population, and the pattern o π indicates the candidate region
under selection [32,33]. Among all 13 populations, the highest π was
ound in RJF (4.90 × 103), and the lowest in RIR (1.74 × 103) (Sup-
plementary le1: Table S4). The commercial breeds, COR, RIR, and
WLH lost 58.78% o their π, which is the same level lost in a previous
study [34]. Among the Korean native chickens, KNR (3.46 × 103) had
the highest π, ollowed by KNW (3.05 × 103), KNG (3.35 × 103), KNB
(3.41 × 103), and KNY (3.45 × 103), and the lowest was observed in
OGYE (3.00 × 103). CNS (3.11 × 103) presented the highest π among
the Chinese indigenous breeds, ollowed by CNT (2.51 × 103) and CNF
(2.42 × 103). The latter two had relatively lower π due to isolated local
adaptation [35] and articial selection or cockghting, respectively
[36]. OGYE had comparatively low levels o π because it is a homoge-
neous breed with small population that approximately 2000 OGYE are
preserving by Korean government conservation program. We also
examined the LD patterns o the population, as they reveal evolutionary
orce, such as genetic bottlenecks and selection signatures [37,38]. We
observed the highest LD level in RIR, ollowed by the rest o the com-
mercial breeds, WLH, and COR, whereas RJF displayed the lowest LD
level (Fig. 3A). Indigenous chicken breeds, such as CNS, OGYE, and
KNC, which showed a moderate level o LD, were classied between RJF
and commercial breeds (Fig. 3A).
3.3. Genome-wide genetic dierentiation o chickens
To quantiy the genetic dierentiation and population structure,
PCA, admixture analysis, phylogenetic tree analysis, and xation index
value (Fst) were perormed. We analyzed 188 chickens rom 13 pop-
ulations that were grouped into our clusters in the neighbor-joining
5
Genomics 114 (2022) 110298
phylogenetic tree. The rst cluster represented nine RJF individuals,
the ancestor o the domestic chicken. The second cluster comprised the
Chinese indigenous breeds o CNF (10), CNT (18), and CNS (31)
chickens. The third cluster involved the commercial breeds o COR (20),
RIR (20), and WLH (20), and the ourth cluster contained the Korean
native chicken breeds (Fig. 2A). The PCA detected the ollowing
phylogenetic relationships. The genetic variances o PC1 and PC2 (PC1:
14.63% and PC2: 11.76%) were distributed to the seven groups
(Fig. 2C). The PCA in Korean native chickens showed that OGYE was
entirely separated rom KNC (PC1: 15.45%, PC2: 11.39%) (Fig. 2D). This
trend was also conrmed in the Fst statistics. OGYE showed high
average levels o genetic dierentiation than the Chinese indigenous
chicken breeds and KNC (Supplementary le1: Table S5). We also
examined the highest levels o Fst in commercial breeds. To determine
the admixture structure across the 188 samples, we perormed admix-
ture analysis with K ranging rom 2 to 10 (Fig. 2B). We observed the best
estimated genetic group separation at K = 7, which showed the same
clustering as the above PCA. The three commercial breeds were sepa-
rated rom K = 2 to K = 5. Furthermore, KNC was distinguished rom
RJF, CNF, CNT, and CNS at K = 4. Admixture analysis measured the
unique genetic admixture o OGYE at K = 6 (Fig. 2B). RJF, CNF, and CNT
ormed a group that mainly represented the ancestral genetic structure
based on admixture analysis. Furthermore, CNS appeared as a mixture o
the ancestral group and KNC. These results support a previous study on
the origin o Korean native chicken breeds introduced into the Korean
Peninsula rom China [39].
3.4. Identifcation o genome-wide selection signature
Genetic change triggers local adaptation when individuals rom the
population have benecial tness traits, such as in morphology cam-
oufage ability, innate immune responses, and etal viability. This leads
to genetic and phenotypic population dierentiation and changes in
genetic structure over time [40]. The peninsula as an island environ-
ment creates geographical isolation and leads to environmental het-
erogeneity, contributing to unique genetic patterns [41]. To identiy the
genomic regions most aected by environmental pressure, we examined
both XP-EHH and XP-CLR, as these methods have previously been suc-
cessully used to detect selection genomic regions in cattle [42], goats
[43], pigs [44], and chickens [36]. In addition, XP-EHH is able to detect
selective genomic regions within a small sample size [45]. Thereore, we
tested the selection analysis o OGYE with a total o 13 chicken breeds;
ancestral (RJF), Chinese indigenous breeds (CNS), Korean native
chicken breeds (OGYE, KNB, KNG, KNR, KNW, and KNY), and com-
mercial breeds (COR, RIR, and WLH) to identiy candidate genes un-
derlying selective pressure or local adaptation. We split the genome
region into overlapping window sizes o 50 kb with a 25 kb step size to
examine genomic regions across populations. With a threshold o the top
0.5% outliers o windows being the selective genomic regions, we
identied a total o 293 candidate genes in terms o XP-CLR and XP-EHH
values (Supplementary le2 and le3). We perormed KEGG pathway
analysis to identiy the biological unction o candidate genes (Supple-
mentary le4) [31]. Among the candidate genes, SPP1 (Secreted Phos-
phoprotein 1), HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A
Member 1), P2RX4 (Purinergic Receptor P2X 4), RNASEL (Ribonuclease
L), BRIP1 (BRCA1 Interacting Protein C-terminal Helicase 1), and TLR4
(Toll-Like Receptor 4), were identied as selection signatures o OGYE
associated with the egg development and innate immune system path-
ways (Table 1). Each selection gene leaves a distinct molecular selection
signal at the genome-wide level, such as a pattern o π , LD, or haplotype
diversity. In terms o the genetic hitchhiking eect, alleles o nearly
linked loci are aected, while an allele o a selection locus is infuenced
by selection pressure. The genomic selection signature leaves a long
haplotype length with low diversity and aects a low crossing over or π
[46]. In addition, the hitchhiking eect results in an intense increase in
LD rom the selected region [33,47]. We scanned or potential genomic
Y. Cho et al. Genomics 114 (2022) 110298

Table 1
Summary o major candidate genes classied rom XP-EHH and XP-CLR in OGYE and KNC.
Selected genes Association CHR XP-CLRa XP-EHHb (log p-value) Candidate SNP position Selected breed Reerencec

SPP1 Eggshell calcication 4 2.57 p.Glu252Lys OGYE [84]

P2RX4 Eggshell calcication 15 2.89 OGYE [54]
RNASEL Innate immune system 8 5.50 p.Asp594Asn OGYE [85]
BRIP1 Fanconi anemia 19 413.76 2.82 OGYE [86]
HSP90AA1 Egg development & Heat resistance 5 6.79 OGYE [87]
TLR4 Innate immune system 17 3.52 p.Gly225Glu OGYE/KOR [88]
NFKBIA Salmonella inection 5 2.56 KOR [73]
DYNC1H1 Salmonella inection 5 2.72 KOR [71]
a
Candidate genes associated with selection signatures, belonging to the top 0.5% o the empirical distributions o XP-CLR results.
b
Candidate genes associated with selection signatures, belonging to the top 0.5% o the empirical distributions o XP-EHH results.
c
Reerences to candidate genes reported related to selection signature.

selection regions based on: (I) low π and haplotype diversity; and (II)
high genomic LD region. The patterns o π, LD, and haplotype diversity
were calculated (Fig. 4A-C and Fig. 5A). We investigated a missense
variant inside the candidate gene region under selection and surrounded
14 variants to classiy haplotype structures and requencies (Fig. 4B-D
and Fig. 5B). In addition, long haplotype length was measured utilizing
the degree o haplotype-sharing by visualizing the major and minor al-
leles o the gene region variant as red and yellow (Fig. 4A-C and Fig. 5A).
Long haplotype length and low haplotype diversity indicated variant
sections that did not combine with the dierent colors. Among the
candidate genes o OGYE, we detected SPP1, RNASEL, and TLR4 genes
that had strong selection signals through rigorous selection analysis
(Fig. 4 and Fig. 5).
3.5. The selection signature o OGYE to the egg development
Previous experimental studies have reported that OGYE has the
heaviest egg with the thickest shell among the Korean native chicken
breeds [11,48]. The quality o eggs is a crucial actor in the viability o
the chick embryo and the ultimate hatchability [49]. We identied a
selection signature or OGYE in the SPP1, P2RX4, and HSP90AA1 gene
regions. We conrmed that SPP1 and P2RX4 are prominent eggshell
matrix protein genes in both the bone and eggshell [50,51]. SPP1 is
highly expressed in the uterine wall during eggshell calcication [52].
The P2RX4 gene plays an important role in calcium signaling and ion
transer pathways or eggshell ormation [53,54], and has been reported
as associated with bone mineral density in a human osteoporosis risk
study [53]. Heat shock proteins (HSPs) reinorce the primary deense
against heat stress [55,56], as they are a group o specic proteins

Fig. 4. Selection signature associated with egg development. (A) Plots o the π, Average LD, and haplotype diversity at the SPP1 region (top) and the pattern o
haplotype sharing among 13 chicken breeds (bottom) in SPP1 region. (B) Gene (top) and haplotype structure (bottom) or the region, including one missense variant
in SPP1 region. The missense variants are located 45,974,845 on chromosome 4.

6
Y. Cho et al. Genomics 114 (2022) 110298

Fig. 5. Selection signature associated with innate immune response. (A,C) Plots o the π, Average LD, and haplotype diversity at the RNASEL and TLR4 regions (top),
and the pattern o haplotype sharing among 13 chicken breeds (bottom) in RNASEL and TLR4 regions. (B–D) Gene (top) and haplotype structure (bottom) or the
region, including one missense variant in RNASEL and TLR4 regions. The missense variants are located at 5,626,065 on chromosome 8 or RNASEL and 4,085,707 on
chromosome 17 or TLR4.

7
Y. Cho et al.
synthesized in response to heat stress. In stressed cells, HSPs bind to
heat-sensitive proteins to protect cells, and the expression rates o HSPs
increase in response to stressul external environments and heat
[57–59]. HSP90AA1 interacts with proteins in older olding stages, as
well as modies the congurations o HSP proteins [60]. Environmental
stress is detrimental to body weight, leading to a decrease in voluntary
eed intake [61]. Egg production, as well as egg weight and eggshell
quality, is attenuated by environmental stress via reproductive hormone
levels and intestinal calcium uptake [62,63]. The most signicant
concern o stressors is the decrease in embryo development in poultry
production [64]. In particular, one missense variant was located at
45,974,845 bp position (Glu252Lys) in SPP1 (Fig. 4D) and a high pattern
o LD and low patterns o π and haplotype diversity were observed. The
spotless haplotype sharing pattern o SPP1 was characterized rom other
chicken breeds (Fig. 4C). These results provide evidence that OGYE has
experienced intense selective pressure, which infuences the allele re-
quency spectrum. We conrmed that SPP1, P2RX4, and HSP90AA1 are
xed genes in OGYE which maintain egg development.
3.6. The selection signature o OGYE and KNC to the innate immune
system
A previous OGYE immunological study has reported decreased nitric
oxide (NO) levels and pro-infammatory cytokines. Three regions o
OGYE (bone, meat, and rind) attenuated the production o NO and pro-
infammatory cytokines in LPS-induced RAW macrophage-derived
264.7 cells. In previous study, termination o infammation was noted
in OGYE. Macrophages are important immune cells or evoking
infammation against harmul stimuli via NO and pro-infammatory
cytokines. However, the termination o infammation is a crucial step
in preparing or other assaults. OGYE might have the ability to increase
anti-infammatory eects o the innate immune system [65]. RNASEL is
the activator gene o the innate immune system that provides a rapid
protective response against viral inection through the initiation o other
immune-related genes [66]. We identied the HSP90AA1 and BRIP1
genes associated with the Fanconi anemia pathway, and Fanconi anemia
is a clinical sign o the immune response [67]. Fanconi anemia is a
recessively derived chromosomal instability disorder. The DNA repair
gene BRIP1 is essential or crosslink resistance and is thereore related to
the Fanconi anemia pathway [68]. TLR4 is an innate immune pattern
recognition receptor that can detect gram-negative bacteria and plays an
essential role in innate and adaptive immune responses [69]. Notably,
we classied the gene regions or missense variants in RNASEL and
TLR4, and both genes showed a high pattern o LD, low patterns o π and
haplotype diversity, and a simple haplotype sharing pattern (Fig. 5A-B).
The missense variants were located at the 5,626,065 bp position
(Asp2Asn) in RNASEL and 4,085,707 bp position (Gly2Glu) in TLR4.
HSP90AA1, BRIP1, and TLR4 contribute to the biological response to
environmental adaptation and the robust innate immune system to anti-
biotic activity. Based on these results, we propose that OGYE has
accumulated environmental pressures and has a strong innate immune
system.
We also identied the KNC selection genomic regions, since KNC has
previously reported strong environmental stress resistance and a high
survival rate [70]. We identied TLR4, NFKBIA (NFKB Inhibitor Alpha),
and DYNC1H1 (Dynein Cytoplasmic 1 Heavy Chain 1) as candidate
genes associated with antibiotic and viral inections. TLR4 is addition-
ally selected on KNC, which is critical or the innate immune system.
Selection analysis o TLR4 showed a high LD, and low π and haplotype
diversity patterns (Fig. 5C). DYNC1H1 directly interacts with the poly-
merase acidic protein o the Infuenza A virus, and plays a crucial role in
the dynein complex, which is related to replication o the H5N1 infu-
enza virus [71]. The NFKBIA gene is also critically involved in the
process that regulates the innate immune system [72]. In a previous
study, the NFKBIA gene induced an immune response to Salmonella
inection in chicken macrophages, which play an anti-infammatory role
8
Genomics 114 (2022) 110298
in both innate and adaptive immunity [73]. The selection signature o
KNC conrmed that it is crucial or the innate immune system unc-
tioning and biotic inection resistance in local adaptation on the Korean
Peninsula.
4. Discussion
The genetic resources o indigenous animals are crucial or biodi-
versity conservation, as they have an excellent ability to adapt to new
environmental conditions [74]. However, many indigenous animals are
declining due to degraded environments and lack o commercial value
[75]. The importance o genetic resources rom indigenous livestock is a
global matter. Many previous studies on indigenous animals have re-
ported genetic characteristics and selection signatures by WGS data:
cattle [42], goats [43], and chickens [36]. WGS is a powerul resource
that captures genetic variation at a high genome resolution with high
accuracy and is superior to the imputation o rare variants [76,77].
This study is the rst OGYE study that ocuses on revealing the se-
lection signature o OGYE and identiying their distinct genomic char-
acteristics using WGS data. OGYE is a representative Korean native
chicken that has long existed on the Korean Peninsula. According to the
Korean traditional medical document ‘Donui Bogam’ (1613), edited by
the royal physician Heo Jun, OGYE was identied as a medication or
human disease [78]. This breed is identied as the 265th Natural Korean
Monument, and is only allowed to be raised in the Gyeryong Mountain
Region (South Chungcheong Province, South Korea) or the conserva-
tion o their phenotypic and genetic characteristics. We utilized a WGS
(16.73×) o over 188 samples rom 13 chicken breeds, including
ancestral breeds, indigenous breeds, and commercial breeds. This study
identied 42,993,594 raw variants and 18,902,359 bi-allelic SNP vari-
ants on 28 autosomes o 13 chicken breeds by WGS data. Previous
genomic OGYE studies mainly used microsatellite and mitochondrial
DNA (mtDNA) sequencing data to evaluate genetic characteristics
[70,79–81]. In a previous OGYE study with mtDNA, a 1231–1232 bp
DNA ragment rom the mtDNA displacement-loop (D-loop) region was
sequenced and 35 variable sites that classied into 21 haplotypes were
detected [39]. WGS helps to overcome the potential misidentication in
analysis and identiy the genetic ootprints o the high-resolution
genome. This study conrmed the benet o WGS in research on ge-
netic characteristics and enhanced the picture o previous microsatellite
and mtDNA studies.
We employed several population genetic methods to identiy novel
genomic characteristics o OGYE by analyzing the genome statistics
summary and comparing empirical genomic distributions to identiy
outliers, which are considered as selection signatures. OGYE exhibited
the lowest π and an intermediate level o LD among indigenous breeds
(Fig. 3A and Supplementary le 1: Table S3). The π level indicates a
large number o homozygous SNP variants dierentiated rom other
indigenous breeds. Moreover, the level o LD observed compared to
other indigenous breeds and emphasized the importance o genetic di-
versity conservation. Furthermore, the PCA results separated OGYE to
the rightmost and unique genetic structure in the admixture analysis
(Fig. 2B-D). The genomic structure o CNS is composed o an ancestral
breed (Red) and KNC (light green) components (Fig. 2B). China is known
as the region o the rst domesticated chicken habitat that existed
approximately 10,000 years ago [6]. In the historical record or KNC,
chicken eggs were discovered in the ancient tomb o Cheonmachong
(Gyeongju, South Korea), which dates to 400 to 500 CE, and KNC
originated rom Chinese ancestors through more than ve maternal lines
based on the mtDNA D-loop variation analysis [39]. The variation o
eective population size over time is illustrated by SMC++, which has
recently been developed or inerring the size o the history o the
population using WGS data (Fig. 3B). OGYE and KNC experienced a
severe population decline approximately 2000 years ago. This result is
compatible with the rst population bottleneck ollowing domestic
chicken arrival and local adaptation on the Korean Peninsula [5]. The
Y. Cho et al.
eective population size o OGYE and KNC slightly increased ater
massive decay due to successul adaptation on the Korean Peninsula.
These previous studies indicated that KNC was introduced rom China or
Southeast Asia into the Korean Peninsula approximately 2000 years ago
[5]. We also observed a linear directional relationship o ancestral (RJF)
and indigenous breeds (CNS and KNC) in PCA, representing migration o
domestic chickens (Fig. 2D). Furthermore, CNS has maintained a spe-
cic genetic admixture o ancestral breeds since domestication, and the
indigenous genetic portion o CNS genetic admixture has been xed on
KNC (Fig. 2B). We conrmed that the OGYE and KNC experienced a
genetic bottleneck event 2000 years ago, when introduced on the
Korean Peninsula, by analyzing the eective population size (Fig. 3B).
These results support the previous study on the origin o the KNC. Based
on these results, OGYE and KNC have mediated local adaptation to
environmental pressures over a long period and have ormed their
unique genomic characteristics by genetic isolation on the Korean
Peninsula.
This study identied that OGYE established selection signatures or
egg development and innate immune system pathways (Table 1). Egg
orms a natural physical barrier to protect the chick embryo and has
mechanical properties that allow the interaction o mineral elements on
the external surace o the eggshell membranes [82]. Newly hatched
chicks are exposed to several pathogenic viruses and microbes, and their
adaptive immune system has a limited ability to deend against these
pathogens. Innate immune systems are crucial or synergizing with
adaptive immune systems to recognize oreign antigens and eliminate
them rom the body [83]. Both egg development and the innate immune
system are essential components o health-related traits in chicken, such
as disease prevention and etal viability, which can be aided by unrav-
eling the genetic basis o the local adaptation to the Korean Peninsula. In
particular, the innate immune system pathway was identied in the
selection signature o KNC (Table 1). Selection signature or the innate
immune response to Salmonella inection in Korean native goats has
been previously reported using whole-genome based evidence [43]. We
proposed that OGYE has selection signatures on egg development and
the innate immune system to protect rom viral inection and pathogenic
microbes, acilitating their local adaptation to the Korean Peninsula
environment.
5. Conclusion
In conclusion, we report, or the rst time, a catalog o selected ge-
netic variants detected in OGYE. We analyzed the π, LD pattern,
phylogenetic tree, genetic dierentiation, admixture, and genome-wide
selection analysis across 188 chicken samples rom 13 populations. This
study determined the unique genetic characteristics o OGYE, which
were dierentiated rom other chicken breeds. Importantly, we identi-
ed the selection signature o OGYE, the RNASEL, BRIP1, and TLR4 gene
regions as essential or promoting the innate immune system, with the
SPP1, P2RX4, and HSP90AA1 genes involved in egg development. These
results could provide targeted genomic characteristics or in vitro and in
vivo studies to analyze advanced hypotheses rom this study and identiy
the underlying genomic mechanisms. Together, these results generate
novel insights into the chicken breeding program or risk and vulnera-
bility management through indigenous animal genetic resources and
acilitate the conservation o OGYE.
Ethics statement
This study was conducted ollowing the Institutional Animal Care
and Use Committee (IACUC) guidelines and was approved by the Na-
tional Institute o Animal Science, Rural Development Administration,
Republic o Korea (Approval No: 2012-D-010). All animal experimen-
tation were conducted under the protocol approved by the guidance o
the IACUC.
9
Genomics 114 (2022) 110298
Funding
This research was supported by the Korea Research Institute o
Bioscience and Biotechnology Research Initiative Program
(1711134074), Ministry o Education (2020M3A9D8038194), and Na-
tional Research Foundation o Korea (2014M3C9A3064552).
Data availability statement
The dataset generated or this study can be ound in the ENA Browser
project name: PRJEB44919 2021-05-15.
Declaration of Competing Interest
None.
Appendix A. Supplementary data
Supplementary data to this article can be ound online at https://doi.
org/10.1016/j.ygeno.2022.110298.
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