GENETICS AND GENOMICS

Molecular phylogenetic analysis of Chinese indigenous blue-shelled chickens

inferred from whole genomic region of the SLCO1B3 gene

Seyed Benyamin Dalirsefat, Xianggui Dong, and Xuemei Deng1

National Engineering Laboratory for Animal Breeding and Key Laboratory of Animal Genetics, Breeding, and
Reproduction of the Ministry of Agriculture, China Agricultural University, Beijing 100193, China

ABSTRACT In total, 246 individuals from 8 Chinese
indigenous blue- and brown-shelled chicken populations
(Yimeng Blue, Wulong Blue, Lindian Blue, Dongxiang
Blue, Lushi Blue, Jingmen Blue, Dongxiang Brown, and
Lushi Brown) were genotyped for 21 SNP markers from
the SLCO1B3 gene to evaluate phylogenetic relation-
ships. As a representative of nonblue-shelled breeds,
White Leghorn was included in the study for refer-
ence. A high proportion of SNP polymorphism was ob-
served in Chinese chicken populations, ranging from
89% in Jingmen Blue to 100% in most populations,
with a mean of 95% across all populations. The White
Leghorn breed showed the lowest polymorphism, ac-
counting for 43% of total SNPs. The mean expected
heterozygosity varied from 0.11 in Dongxiang Blue to
0.46 in Yimeng Blue. Analysis of molecular variation
(AMOVA) for 2 groups of Chinese chickens based on
eggshell color type revealed 52% within-group and 43%
Key words: blue-shelled chicken, SLCO1B3 gene, EAV–HP insertion, SNP marker, phylogenetic analysis
between-group variations of the total genetic variation.
As expected, FST and Reynolds’ genetic distance were
greatest between White Leghorn and Chinese chicken
populations, with average values of 0.40 and 0.55, re-
spectively. The first and second principal coordinates
explained approximately 92% of the total variation and
supported the clustering of the populations according to
their eggshell color type and historical origins. STRUC-
TURE analysis showed a considerable source of vari-
ation among populations for the clustering into blue-
shelled and nonblue-shelled chicken populations. The
low estimation of genetic differentiation (FST ) between
Chinese chicken populations is possibly due to a com-
mon historical origin and high gene flow. Remarkably
similar population classifications were obtained with all
methods used in the study. Aligning endogenous avian
retroviral (EAV)–HP insertion sequences showed no dif-
ference among the blue-shelled chickens.
2015 Poultry Science 94:1776–1786
http://dx.doi.org/10.3382/ps/pev146

INTRODUCTION phenotypic quality to their mates. Morales et al.

Eggshell color is both of biological interest and of
economic importance, and the nature of the shell pig-
ments in various avian species and biochemical and
physiological processes involved in pigment formation
and its deposition in and on the shell have been dis-
cussed since the 19th century (Wicke, 1858; Sorby, 1875;
Krukenberg, 1883; Lang and Wells, 1987). Because it is
an important aspect of egg quality in many countries,
shell color has been emphasized in the sales, promotion,
and marketing strategies of egg retailers.
Recently, avian egg color has been explained as
mainly serving crypsis or mimetism (Underwood and
Sealy, 2002). Blue–green colors in avian eggs have
also been proposed as post-mating signals of female

C 2015 Poultry Science Association Inc.
Received December 6, 2014.
Accepted April 14, 2015.
1
Corresponding author: deng@cau.edu.cn
(2006) suggested that egg color may not only indicate
female value, but also the quality of the eggs them-
selves and of the resulting offspring. Blue–green eggshell
color in chickens is produced by an autosomal dominant
trait called oocyan, and eggs laid by oocyan homozy-
gotes are a darker blue than those from heterozygotes
(Punnett, 1933).
SLCO1B3 codes a membrane transporter OATP1B3,
which is considered a liver specific transporter and is
highly expressed in liver, where it transports a wide
range of substrates, including bile salts (Popovic et al.,
2010; Hagenbuch and Gui, 2008). Wang et al. (2013)
suggested that, as blue eggshell is colored mainly by
deposition of biliverdin, which is one component of bile
salts, expression of SLCO1B3 in the uterus could en-
hance transportation of biliverdin to the eggshell. They
also found that SLCO1B3 is expressed in the shell
gland of blue-shelled chickens and not in the glands of
brown- or white-shelled chickens, supporting an es-
sential role of the gene in pigmentation of blue eggs.

1776
 PHYLOGENETIC ANALYSIS OF CHINESE CHICKENS
Figure 1. Geographical locations of the 6 indigenous chicken populations sampled in China
Recently, in 2 independent studies, an endogenous avian
retroviral (EAV)–HP insertion has been discovered
in 2 Chinese (Dongxiang and Lushi), an American
(Mapuche fowl), and a European (Araucana) blue-
shelled chicken breeds, associated with the over-
expression of SLCO1B3, strongly suggesting this is a
causative mutation for the blue eggshell phenotype
(Wang et al., 2013; Wragg et al., 2013). In addition,
conserved EAV–HP integration sites and sequences
have been found in South American and European
blue-shelled chickens, distinct from those of the Asian
chicken, which implies independent integration events
in the blue-shelled chickens from the 2 groups of the 3
continents and provides an example of parallel evolu-
tion at the molecular level.
China has a wide variety of indigenous poultry, with
108 native chicken breeds (Chen et al., 2004), many
of which have valuable genetic features. Most of these
chickens are local and fancy breeds characterized by
medium to low performance and are usually maintained
in small populations. The native chicken varieties in-
clude some blue-shelled breeds; Dongxiang and Lushi
are 2 indigenous blue-shelled chicken breeds reported
in recent studies (Zhao et al., 2006; Wang et al., 2013).
However, the origin of some local blue-shelled chickens
in China remains unclear.
Phylogenetic analysis and breed characterization re-
quires basic knowledge of genetic variations that can
be effectively measured within and between popula-
tions. In recent years, analysis of SNP markers has
become the standard approach for diversity analy-
sis and genome-wide studies. Because of their abun-
dance in the genome, genetic stability, and amenity
to high-throughput automated analysis, SNP represent
1777
one of the more interesting approaches for genotyping
(Vignal et al., 2002). The usefulness of SNP in analyses
of population diversity and structure has been demon-
strated in several studies (McKay et al., 2008; Lin et al.,
2010; Edea et al., 2013). Furthermore, with the progress
of sequencing technology, whole-genome/gene sequenc-
ing has become available for characterizing genetic
diversity among farm animals (Yang et al., 2013). This
is an improvement on using microsatellites and microar-
rays because it can detect both SNP and structural
variations. The present study was undertaken to an-
alyze the molecular phylogenetic analysis of 8 Chinese
indigenous blue- and brown-shelled chickens by using
SNP markers inferred from resequencing the whole ge-
nomic region of SLCO1B3 and its inserted EAV–HP
sequence information.
MATERIALS AND METHODS
Sample Collection and DNA Extraction
Eight Chinese indigenous blue- and brown-shelled
chicken populations were used in the study. All sam-
ples were collected from breed conservation farms in the
related regions in China (Figure 1). The breeds were Yi-
meng Blue from Shandong Province (N = 45), Wulong
Blue (N = 45) from Chongqing Municipality, Lindian
Blue (N = 40) from Heilongjiang Province, Jingmen
Blue (N = 42) from Hubei Province, Dongxiang Blue
(N = 28) and Dongxiang Brown (N = 29) from Jiangxi
Province, and Lushi Blue (N = 29) and Lushi Brown
(N = 30) from Henan Province. As a representative of
a nonblue and brown eggshell breed, White Leghorn
(N = 30) was also included for reference. Although the
1778 DALIRSEFAT ET AL.
populations are termed as blue-shelled chicken breeds,
the eggshell color trait has not been fixed. Histor-
ically, Dongxiang and Lushi chickens have been se-
lected for blue eggshell. The other populations have
not been systematically bred until now, so some appear-
ance traits, eggshell color, and feather color do not show
homogeneity.
Total genomic DNA was extracted from blood using
a TIANamp blood DNA kit. The DNA concentration
and its purity were examined using spectrophotometric
analysis based on absorbance at 260 and 280 nm and
agarose gel electrophoresis analysis.
Diagnostic Genotyping Test of EAV–HP
Insertion
The retrovirus insertion was genotyped by multi-
plex PCR using 3 primers (test-nor-up, test-no-down,
and test-eav) introduced by Wang et al. (2013). The
PCR amplifications were performed in a total volume
of 20 μL containing 10 μL 2XTaq PCRMix (TIANGEN
Biotech Beijing Co., Ltd.), 0.25 μM each primer, and
50 to 100 ng genomic DNA in the following conditions:
95◦ C for 5 min, followed by 36 cycles of 95◦ C for 30
s, 56◦ C for 30 s, 72◦ C for 20 s, and a final extension
at 72◦ C for 5 min. The PCR products were separated
by 2% agarose gel electrophoresis, and the length of
target fragments were 340 bp for test-nor-up and test-
nor-down, and 425 bp for test-nor-up and test-eav.
Resequencing of EAV–HP Insertion
The EAV–HP insertion (approximately 4.2 kb) up-
stream of SLCO1B3 was amplified using the method
described by Wang et al. (2013). With the exception
of the Dongxiang Blue, which has been previously se-
quenced by Wang et al. (2013; GenBank Accession
No.: JF837512), we sequenced the EAV–HP insertion
of all blue-shelled chicken populations (GenBank Acces-
sion No.: KP256532, KP276576, KP276577, KP276578,
and KP276579 for Yimeng Blue, Wulong Blue, Lindian
Blue, Lushi Blue, and Jingmen Blue, respectively).
MassARRAY Analysis
Twenty-one SNPs found by resequencing of
SLCO1B3 (Wang et al., 2013) were used to analyze
the genetic variants of SLCO1B3 in the 9 populations.
SNP markers were genotyped using an iPLEX SE-
QUENOM MassARRAY platform (Sequenom, CA).
This genotyping system uses single-base extension
reactions to create allele-specific products that are
separated automatically and scored in a matrix-
assisted laser desorption ionization/time-of-flight mass
spectrometer. Primer design was performed using Mas-
sARRAY Assay Design software (v3.1) according to
Sequenom’s instructions. Multiplex PCR amplification
of amplicons containing SNP of interest was performed
using HotStart Taq Polymerase (Qiagen, CA) with
12 ng genomic DNA. Assay data were analyzed using
Sequenom TYPER software (v3.4).
Statistical Analysis
Genetic diversity Total number of alleles, number
of effective alleles, allele frequencies, observed (Ho) and
expected (He) heterozygosity (Nei, 1973), Shannon’s in-
formation index, and fixation index for each popula-
tion across the loci were estimated using the programs
Arlequin (Excoffier et al., 2005) and GenAlEx 6.501
(Peakall and Smouse, 2012). The fixation index F (also
called the inbreeding coefficient) can take values rang-
ing from −1 to +1. Values close to zero are expected
under random mating, while substantial positive values
indicate inbreeding or undetected null alleles. Negative
values indicate excess of heterozygosity due to nega-
tive assortative mating or selection for heterozygotes.
Deviation from Hardy–Weinberg equilibrium (HWE;
heterozygote deficiency) was assessed by performing a
chi-square test with the PowerMarker program (Liu and
Muse, 2005) for each marker and population.
Analyses of molecular variance Analyses of
molecular variance (AMOVA) were carried out on 3
data sets using the program GenAlEx 6.501 (Peakall
and Smouse, 2012). The first analysis included data
only for 8 Chinese chicken populations in one group;
the second data set consisted of the Chinese chickens
grouped by the 2 eggshell color types, and a third anal-
ysis was performed for all 9 populations in three groups
(i.e., 6 Chinese blue eggshell, 2 Chinese brown eggshell,
and White Leghorn chicken populations).
Genetic differentiation The Wright (1978) fixa-
tion indices were estimated according to Weir and
Cockerham (1984) using the program GenAlEx 6.501
(Peakall and Smouse, 2012). The significance of fixation
indices and pairwise population differentiation values
were determined by permutation tests with 1,000 per-
mutations.
Relationships among the populations Reynolds’
genetic distance (Reynolds et al., 1983) between differ-
ent pairs of chicken populations was calculated using
PowerMarker (Liu and Muse, 2005). The unweighted
pair-group method with arithmetic mean (UPGMA;
Sneath and Sokal, 1973) algorithm was used to con-
struct the dendrogram from Reynolds’ matrices using
the same software. The generated tree was visualized
in Mega tree explorer (Tamura et al., 2011). Gene flow
between populations, defined as the number of repro-
ductively successful migrants per generation (Nm), was
calculated using the program GenAlEx 6.501 (Peakall
and Smouse, 2012). The estimate was based on the re-
lationship FST = 1/(4Nm + 1), where N is the effective
population size, m is the migration rate, and FST is cal-
culated as the mean over loci.
Principal coordinate analysis A principal coordi-
nate analysis (PCoA) was carried out to illustrate the
relationships among the populations using the program
GenAlEx 6.501 (Peakall and Smouse, 2012) based on
 PHYLOGENETIC ANALYSIS OF CHINESE CHICKENS 1779
Table 1. Genetic variability within chicken populations1 .

SNP (%) not

Polymorphic in HWE
Population N Na (SE) Ne (SE) Hob (SE) Hex (SE) I (SE) markers (%) (P ≤ 0.05) F (SE)
Yimeng Blue 45 2.0 (0.00) 1.9 (0.03) 0.75 (0.03) 0.46 (0.01) 0.65 (0.01) 100 95 −0.61 (0.04)
Wulong Blue 45 2.0 (0.00) 1.5 (0.03) 0.39 (0.03) 0.31 (0.02) 0.48 (0.03) 100 29 −0.25 (0.02)
Lindian Blue 40 2.0 (0.00) 1.8 (0.03) 0.72 (0.03) 0.45 (0.01) 0.64 (0.01) 100 90 −0.58 (0.04)
Dongxiang Blue 28 1.9 (0.07) 1.1 (0.01) 0.12 (0.01) 0.11 (0.01) 0.21 (0.02) 90 0 −0.07 (0.00)
Dongxiang Brown 29 1.9 (0.07) 1.3 (0.04) 0.07 (0.02) 0.22 (0.02) 0.37 (0.03) 90 84 0.75 (0.05)
Lushi Blue 29 2.0 (0.05) 1.3 (0.03) 0.30 (0.02) 0.25 (0.02) 0.40 (0.03) 95 0 −0.19 (0.01)
Lushi Brown 30 2.0 (0.00) 1.4 (0.08) 0.21 (0.04) 0.23 (0.04) 0.36 (0.05) 100 19 0.11 (0.07)
Jingmen Blue 42 1.9 (0.08) 1.3 (0.03) 0.27 (0.02) 0.22 (0.02) 0.37 (0.03) 86 0 −0.18 (0.00)
White Leghorn 30 1.4 (0.11) 1.4 (0.10) 0.15 (0.04) 0.19 (0.05) 0.27 (0.07) 43 0 0.22 (0.01)
Mean 1.9 (0.06) 1.4 (0.08) 0.33 (0.02) 0.27 (0.01) 0.42 (0.02) 89 35 −0.12 (0.03)
1
Na = No. different alleles, Ne = No. effective alleles, Hob = Observed heterozygosity, Hex = Expected heterozygosity, I = Shannon’s
information index, and F = Fixation index.

an algorithm published by Orlóci (1978). Two methods
of PCoA were carried out to determine population re-
lationships based on the FST matrix and the covariance
matrix, with data standardization by using a multivari-
ate technique that allows one to find and plot the major
patterns within a multivariate data set (e.g., multiple
loci and multiple samples).
Structure analysis For the analysis of popula-
tion structure, a model-based clustering method us-
ing genotype data consisting of unlinked markers was
implemented with the program STRUCTURE 2.3.4
(Pritchard et al., 2000, 2010). This program can de-
termine the existence of population structure, ascer-
tain distinct genetic populations, assign individuals to
populations, and identify migrants and admixed indi-
viduals. It is assumed that within populations the loci
are at HWE and linkage equilibrium. In the present
study, a Monte Carlo Markov chain algorithm was used
to estimate allele frequencies in each of the K popula-
tions and the degree of admixture for each individual.
The true number of clusters (K) was inferred by the
program STRUCTURE HARVESTER (Earl and von-
Holdt, 2012) implementing the Evanno (2005) method,
using 10 independent runs with 100,000 iterations fol-
lowing a burn-in period of 20,000, under an admixture
ancestry and correlated allele frequency model with K
ranging from 2 to 10. The estimated cluster member-
ship coefficient matrices of multiple runs were used as
the input file for finding optimal alignments of R repli-
cate cluster analyses of the same data using the pro-
gram CLUMPP (Jakobsson and Rosenberg, 2007). The
most frequent solution for each K was taken as the most
probable clustering and visualized using DISTRUCT
software (Rosenberg, 2004).
EAV–HP insertion data analysis Because the
EAV–HP insertion does not occur in White Leghorn, 2
blue-shelled chicken breeds from South America (Chile,
Mapuche fowl) and Europe (France, Araucana) re-
ported by Wragg et al. (2013) were included in this
analysis as outgroups. Multiple alignment of the EAV–
HP insertion sequences of all blue-shelled chickens un-
der study and Mapuche/Araucana (GenBank Accession
No.: KC632578) was done using the online Basic Local
Alignment Search Tool program of the National Center
for Biotechnology Information.
RESULTS
We used fixation indices (FST , FIS , and FIT ) as pri-
mary metrics for empirically estimating and testing
the magnitude of genetic divergence among popula-
tions (Table S1). The fixation coefficients of subpopu-
lations within the total population (FST ) when all pop-
ulations were analyzed for the 21 loci varied from 0.12
(SNP1B3˙4) to 0.54 (SNP1B3˙6), with a mean of 0.41
(P < 0.001). All loci contributed significantly to this
differentiation. The average overall deficit of heterozy-
gotes across populations (FIT ) was 0.29 (P < 0.001) and
the mean FIS was −0.22 (P > 0.05) within populations.
All loci showed significant excess of heterozygotes.
Numbers of different and effective alleles, ratio of
polymorphic markers, Shannon’s information index, ge-
netic variability within the different chicken popula-
tions, fixation index, and departures from HWE are
shown in Table 1. Among Chinese chicken populations,
the proportion of polymorphic SNP ranged from 86% in
Jingmen Blue to 100% in Yimeng Blue, Wulong Blue,
Lindian Blue, and Lushi Brown, with a mean of 95%.
The White Leghorn breed showed the lowest proportion
of polymorphism (43% polymorphic loci). The mean ex-
pected heterozygosity over all loci for each population
varied from 0.11 (0.01) in Dongxiang Blue to 0.46 (0.01)
in Yimeng Blue. The highest observed heterozygosity
was similarly found in the same population (0.75 ± 0.03
in Yimeng Blue), whereas Dongxiang Brown showed
the lowest observed heterozygosity (0.07 ± 0.02). The
mean values of observed and expected heterozygosities
in White Leghorn were 0.15 (0.04) and 0.19 (0.05), re-
spectively. Three populations showed an overall sig-
nificant deficit of heterozygotes, while 6 populations
showed an excess of heterozygous genotypes with re-
spect to the expected value. Effective number of alle-
les and Shannon’s information index ranged from 1.1
(0.01) and 0.21 (0.02) in Dongxiang Blue to 1.9 (0.03)
and 0.65 (0.01) in Yimeng Blue, respectively. The high-
est and the least values of fixation index (F; inbreeding
1780 DALIRSEFAT ET AL.

Table 2. Analysis of molecular variation (AMOVA) analysis results using different data sets of Chinese chicken populations
and White Leghorn.

Data set Variance component (%)

Among population
Among groups Among populations within groups Within groups
Only Chinese chickens in one group − 29 − 71
Chinese chickens based on 2 eggshell 43 − 5 52
color groups
All 9 populations in 3 groups
(Chinese Blue eggshell, Brown eggshell, 44 − 5 51
and White Leghorn group)

Table 3. Pairwise genetic differentiation (FST ) and gene flow (Nm; in parenthesis) values between the 9 chicken populations
(below diagonal) and Reynolds’ genetic distance (above diagonal).

Population Yimeng Wulong Lindian Dongxiang Dongxiang Lushi Lushi Jingmen White
Blue Blue Blue Blue Brown Blue Brown Blue Leghorn
Yimeng Blue 0.088 0.008 0.28 0.35 0.15 0.37 0.17 0.42
Wulong Blue 0.0461 0.073 0.095 0.57 0.023 0.60 0.03 0.64
(5.2)
Lindian Blue 0.004 0.0381 0.26 0.39 0.13 0.41 0.15 0.47
(64) (6.4)
Dongxiang Blue 0.161 0.051 0.151 0.76 0.05 0.78 0.041 0.81
(1.3) (4.8) (1.5)
Dongxiang Brown 0.211 0.401 0.241 0.621 0.64 0.16 0.66 0.37
(0.95) (0.38) (0.78) (0.16)
Lushi Blue 0.081 0.012 0.071 0.025 0.471 0.67 0.002 0.70
(2.9) (21) (3.4) (9.6) (0.29)
Lushi Brown 0.231 0.431 0.261 0.651 0.0841 0.50 1
0.69 0.24
(0.85) (0.33) (0.72) (0.14) (2.7) (0.25)
Jingmen Blue 0.0921 0.016 0.0811 0.021 0.491 0.001 0.531 0.72
(2.5) (15) (2.8) (12) (0.26) (340) (0.23)
White Leghorn 0.271 0.471 0.301 0.681 0.231 0.541 0.141 0.56 1

(0.69) (0.29) (0.58) (0.12) (0.85) (0.22) (1.6) (0.20)

1
0.001 < P < 0.01, significance levels were obtained after 1,000 permutations.

coefficient) were observed in Dongxiang Brown (0.75 ±
0.05) and Yimeng Blue (−0.61 ± 0.04), respectively,
with an average of −0.12 (0.03) across all populations.
All polymorphic SNP were in HWE (P ≤ 0.05) in
Dongxiang Blue, Lushi Blue, Jingmen Blue, and White
Leghorn. However, in the other 5 populations, from 19%
(in Lushi Brown) to 95% (in Yimeng Blue) of SNP
markers significantly deviated from HWE (P ≤ 0.05).
For further analysis, we used AMOVA to examine
the partitioning of genetic variation (Table 2). When
the 8 Chinese chicken populations were analyzed as one
group, most of the population variance (71%) could be
explained by within-group variability. Analysis of the 8
Chinese chicken populations grouped based on eggshell
color types (Chinese blue eggshell and Chinese brown
eggshell) showed that within-group genetic differences
accounted for 52% of the total variation, with 43% vari-
ation among groups. When White Leghorn chickens
were included in the analysis, the percentages of vari-
ance components were very close to the second data
set.
To evaluate genetic differentiation and similarity
among populations, pairwise population FST estimates
and Reynolds’s genetic distance were examined for
each pair of chicken populations (Table 3). For most
pairs, the FST values indicated statistically significant
differences (0.001 < P < 0.01), revealing significantly
differentiated populations. Among Chinese chicken
populations, the lowest FST (0.001) and Reynolds’ ge-
netic distance (0.002) were both found between Lushi
Blue and Jingmen Blue populations, whereas the high-
est values (0.65 and 0.78, respectively) were both found
between Dongxiang Blue and Lushi Brown populations.
As expected, average pairwise FST (0.40) and Reynolds’
genetic distance (0.55) between White Leghorn and
Chinese chicken populations were higher than those
between Chinese chickens, with average values of 0.21
and 0.31, respectively. However, average FST (0.42) and
Reynolds’ genetic distance (0.57) values between all
pairs of blue- and brown-shelled chickens were higher
than those averages between White Leghorn and Chi-
nese chickens (0.40 and 0.55, respectively). Interest-
ingly, when comparing White Leghorn and Chinese
chicken populations, the lowest values of both FST (0.23
and 0.14) and Reynolds’s genetic distance (0.37 and
0.24) were found between White Leghorn and 2 brown-
shelled types of Chinese chickens. However, some pairs
of blue-shelled types did not show significant differen-
tiation (P > 0.05, Table 3).
We also estimated gene flow (Nm) between each pop-
ulation pair (Table 3) to examine the possible move-
ment of individuals and genes in space, which affects
many important ecological and evolutionary properties
of populations. The Nm value across Chinese chickens
 PHYLOGENETIC ANALYSIS OF CHINESE CHICKENS
Lushi Blue
Jingmen Blue
Wulong Blue
Dongxiang Blue
Yimeng Blue
Lindian Blue
White Leghorn
Dongxiang Brown
Lushi Brown
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00
Figure 2. Unweighted pair-group method with arithmetic mean
(UPGMA)-based phylogenic tree showing the genetic relationships
among the 8 Chinese chicken populations and White Leghorn using
Reynolds et al. (1983) genetic distance.
ranged from 0.14 (between Dongxiang Blue and Lushi
Brown) to 340 (between Lushi Blue and Jingmen Blue).
Between Chinese chickens and White Leghorn, Nm var-
ied from 0.12 to 1.6. As expected, the mean number
of migrants per generation (Nm) across all Chinese
chicken populations (18) was much higher than that
between White Leghorn and Chinese chickens (0.56),
while the average estimated gene flow across all popu-
lations was 14. All Nm values between Chinese chicken
populations with the same eggshell color were above
1.0, whereas values between populations with different
eggshell colors were below 1.0. In addition, the Nm val-
ues between White Leghorn and Chinese populations
(except Lushi Brown) were below 1.0.
We also used a tree-structured graph to visualize the
result of hierarchical clustering and the phylogenetic
relationships among the 9 chicken populations, based
on Reynolds’ genetic distance (Reynolds et al., 1983;
Figure 2). As expected, the populations clearly sep-
arated into 2 major clusters, including all blue-
shelled chickens in the first major cluster and brown-
shelled chickens together with White Leghorn in the
second major cluster. Within Chinese blue-shelled
chicken populations, Lindian Blue and Yimeng Blue
formed a closely related subcluster. Lushi Blue and
Jingmen Blue also formed a very closely related sub-
cluster. Dongxiang Blue and Wulong Blue were inter-
Figure 3. First and second principal coordinates analyses (PCoA) results in the 8 Chinese chicken populations and White Leghorn clustered-
based on populations (a) and individuals (b).
1781
mediately positioned between these 2 subclusters. In
the second major cluster, Dongxiang Brown and Lushi
Brown formed a relatively close group separated from
White Leghorn.
Since matrices such as the pairwise FST matrix for
9 populations or 318 individual chickens can be dif-
ficult to read and interpret, PCoA was used to vi-
sualize the patterns of genetic relationship. Analy-
sis of principal coordinates based on the FST matrix
for 21 markers in the 9 chicken populations is illus-
trated in Figure 3(a). PCoA obviously discriminated
blue-shelled chickens from other chicken populations,
with the first and second principal coordinates explain-
ing 83 and 8.7%, respectively, of the total variation.
Figure 3(b) shows PCoA results based on a genetic dis-
tance matrix for 318 individuals and a covariance ma-
trix with data standardization of the 9 chicken pop-
ulations. This PCoA also apparently separated Chi-
nese blue-shelled chickens from brown-shelled chickens
and White Leghorn, with the first and second princi-
pal coordinates explaining 75 and 7.2%, respectively,
of the total variation. The third principal component
accounted for approximately 4.1% of the variation,
and slightly separated the White Leghorn population
from the brown-shelled chickens, with some overlap-
ping. PCoA analysis confirmed the results of the pop-
ulation structure and dendrogram analysis.
The program STRUCTURE was used to investigate
population structure as well as inferring the presence
of distinct populations, assigning individuals to popu-
lations, studying hybrid zones and identifying migrants
and admixed individuals. The graphic results of the
clustering analysis for K = 2 to 7 are illustrated in Fig-
ure 4. Results of the Bayesian clustering approach for
K = 2 to 10 are presented in Table S2 and Figure S1,
and show that K = 7 was optimal, capturing the major
structure proportion present in the data (Figure S2).
With K = 2, most blue-shelled chicken populations
appeared clearly differentiated from other chickens,
and the Yimeng Blue with Lindian Blue populations
showed intermediate results (Figure 4 and Table S3).
At this K value, 7 populations clustered with more
than 76% of membership coefficients, while more than
54% of individuals from Yimeng Blue and Lindian Blue
1782
Figure 4. STRUCTURE clustering of Chinese indigenous blue- and brown-shelled chickens in reference to the White Leghorn. Each individual
is represented by a single vertical segments showing estimated membership coefficients of each individual to the inferred K cluster, with K = 2
to 7. Black color line separated the populations indicated below the figure.
clustered in the related group (data not shown). This
result is in good agreement with results from UPGMA
dendrogram and PCoA. With K = 3, a relatively high
heterogeneity of membership coefficients was observed
within blue-shelled chicken populations, particularly in
comparison with nonblue-shelled chickens. Differenti-
ation within Chinese blue-shelled chicken populations
was first observed at K = 3, with over 50% of Wulong
Blue, Dongxiang Blue, Lushi Blue, and Jingmen Blue
assigned to the same cluster, and 62% of the Yimeng
Blue and 61% of Lindian Blue sharing the same cluster.
However, there was no clear differentiation among blue-
shelled chicken populations at these inferred clusters
and they did not cluster according to their traditional
classifications or geographical distribution.
Similar to K = 3, 3 clusters were formed at K = 4, but
with a lower proportion of individuals in each cluster.
At this K value, White Leghorn as an outgroup chicken
started to differentiate from brown-shelled chickens.
At K = 5 and 6, the same clusters that formed at
K = 4 were observed, with approximately close mem-
bership coefficient. Finally, at K = 7, Wulong Blue and
Dongxiang Blue separated from Lushi Blue and Jing-
men Blue to form different clusters.
Since EAV–HP insertion is the causative mutation
for the blue-shelled phenotype (Wang et al., 2013), we
sequenced and aligned the EAV–HP insertion of the
populations to examine potential polymorphism. Multi-
ple sequence alignment from upstream and downstream
parts of the EAV–HP integration site, corresponding to
the 6 blue-shelled chicken populations (GenBank Acces-
sion No.: KP256532, KP276576, KP276577, KP276578,
KP276579, and JF837512 for Yimeng Blue, Wulong
Blue, Lindian Blue, Lushi Blue, Jingmen Blue, and
Dongxiang Blue, respectively) and Mapuche/Araucana
(GenBank Accession No.: KC632578; Wragg et al.,
DALIRSEFAT ET AL.
2013) as an outgroup, compared with the host sequence,
Gallus gallus OATP1B3 (SLCO1B3) gene (GenBank
Accession No.: JN020139) is illustrated in Figure S3.
Complete sequences of the EAV–HP insertions re-
vealed 100% identity among Chinese blue-shelled chick-
ens except for the Dongxiang breed (GenBank Acces-
sion No.: JF837512; Wang et al., 2013). However, the
EAV–HP sequences of the all oocyan Chinese chickens
were 98% identical to Mapuche/Araucana sequences.
Comparing sequences of the complete insertion in the
homozygote oocyan chickens illustrated identical host
integration sites at Gga1:67,324,647 for all the Chinese
breeds, but a difference at Gga1:67,324,624 for the Ma-
puche/Araucana chickens. In addition, the target-site
duplications were identical in all the Chinese breeds (3 -
GAGGAG-5 ) but different in the Mapuche/Araucana
(3 -CCTTCA-5 ) chickens (Figure S3).
DISCUSSION
We evaluated the molecular phylogenetic relation-
ships among 8 Chinese indigenous blue- and brown-
shelled chickens, with White Leghorn as an outgroup,
by using SNP markers inferred from resequencing the
whole genomic region of SLCO1B3 and its inserted
EAV–HP sequence.
All SNP loci identified in the whole SLCO1B3 gene of
Chinese chicken populations exhibited a high degree of
polymorphism (average 95%), whereas White Leghorn
showed the lowest level (43%; Table 1). Genetic vari-
ability was highest in Yimeng Blue chicken (0.46 ±
0.01), while Dongxiang Blue demonstrated the lowest
genetic variability (0.11 ± 0.01). The relatively higher
genetic diversity observed in the Yimeng Blue popula-
tion and Lindian could be due to lack of inbreeding
(F = −0.61 and −0.58, respectively) and controlled
 PHYLOGENETIC ANALYSIS OF CHINESE CHICKENS
mating practices. The genetic variability of White
Leghorn (0.19 ± 0.05) was lower than for Chinese
chickens except Dongxiang Blue (0.11 ± 0.01). The
lower genetic variability in White Leghorn is in har-
mony with the results of Leroy et al. (2012), who
observed lower genetic diversity in white egg layers
compared with 24 other African local and commercial
breeds. This may correspond to the somewhat wider
genetic range of founder breeds in local chickens than
in white egg layers and to the lower number of breed-
ing generations (Crawford, 1990). The observed het-
erozygosity values were less than expected in Dongxiang
Brown, Lushi Brown, and White Leghorn, causing F >
0, which could indicate an inbreeding system of mat-
ing (Templeton and Read, 1994) in these populations.
In contrast, higher observed heterozygosity than ex-
pected in the remaining populations might indicate an
isolate-breaking effect (opposite of the Wahlund effect).
Among all chicken populations, the average observed
and expected heterozygosity values were lower in this
SNP-based study (Table 1) than when estimated using
microsatellite markers in several studies, including 52
European chicken breeds (Hillel et al., 2003), 12 Chinese
indigenous chicken breeds (Granevitze et al., 2007), 15
Chinese indigenous chicken breeds (Chen et al., 2008),
6 Italian local chicken breeds (Zanetti et al., 2007),
10 Egyptian chicken strains (Eltanany et al., 2011), 5
Japanese native chicken breeds (Tadano et al., 2012), 23
African local breeds (Leroy et al., 2012), and 18 African
native chicken breeds (Berima et al., 2013). By con-
trast, lower values of heterozygosity (0.19 ± 0.02) have
been described for Italian native chickens using Ampli-
fied Fragment Length Polymorphism (AFLP) markers
(De Marchi et al., 2006). The average expected het-
erozygosity within populations (0.27 ± 0.01) in this
study was very close to the value reported for layer
lines (0.27), but lower than for broiler lines (0.53) of
commercial breeds analyzed by using 17 microsatellites
(Crooijmans et al., 1996). However, the observed het-
erozygosity values of Yimeng Blue (0.75 ± 0.03) and
Lindian Blue (0.72 ± 0.03) were substantially higher
than those of all chicken breeds reported in the stud-
ies noted above. The differences in the results may
be explained by differences in locations, sample sizes,
experimental chickens, application of different molec-
ular markers (particularly the multi-allelic nature of
microsatellite markers), and wider distribution of mi-
crosatellite and AFLP markers throughout the genome.
Five populations showed significant deviation from
HWE (P ≤ 0.05) ranging from 19% in Lushi Brown to
95% in Yimeng Blue. This significant violation may be
explained by sampling errors (including the Wahlund
effect), misclassification of genotypes, measuring 2 or
more systems as a single system, population substruc-
ture, failure to detect rare alleles and the inclusion of
nonexisting alleles, or if inbreeding has occurred in the
populations as a whole.
As expected, estimates of pairwise population dif-
ferentiation (FST ) and Reynolds’ genetic distance
1783
(Table 3) revealed closer relationships between Chinese
chicken populations than between White Leghorn and
Chinese chickens. This is in good agreement with an
AFLP-based fingerprinting study (Gao et al., 2008),
which indicated higher genetic similarity among indige-
nous chicken breeds in China compared with that be-
tween Recessive White breed and indigenous Chinese
chicken breeds. A low level of differentiation was also
observed between each pair of the Chinese populations
according to their eggshell color types, which could be
attributed to common ancestry, admixture of the pop-
ulation, and lack of selection pressure in these pop-
ulations. On the other hand, the average FST among
Chinese populations across loci observed in this study
(0.35; Table S1) was higher than that reported between
5 closely related lines of Japanese native chickens (FST
= 0.15; Tadano et al., 2012), 15 Chinese indigenous
chicken breeds (FST = 0.16; Chen et al., 2008), and
78 Chinese indigenous chicken breeds (FST = 0.11, Qu
et al., 2006), but lower than that between local Italian
breeds of chickens (FST = 0.44; Zanetti et al., 2007).
However, the range of pairwise FST between all popula-
tions in the present study (from 0.001 to 0.68; Table 3)
was more extensive than that between 5 closely related
lines of Japanese native chickens, the Nagoya breed
(from 0.022 to 0.25), assessed based on microsatellite
polymorphisms (Tadano et al., 2012).
The average FIS value over loci (−0.22 ± 0.02; Table
S1), which indicates the degree of departure from ran-
dom mating, was significantly negative in this study,
similar to that of all commercial crosses and 2 Nagoya
lines of Japanese native chickens (−0.14; Tadano et al.,
2012), reflecting excess heterozygosity. In contrast, Ital-
ian chicken breeds (0.042; Zanetti et al., 2007) and 15
Chinese indigenous chicken breeds (0.02; Chen et al.,
2008) had positive and higher values of FIS , indicating
heterozygosity deficiency and departures from random
mating as a result of inbreeding within populations. As
Edea et al. (2013) suggested, the absence of any sig-
nificant inbreeding effects may be a reflection of the
high gene flow between the populations, as supported
by high Nm values (Table S1), the large population from
which the samples were drawn, and the fact that related
individuals were purposely avoided.
In AMOVA analysis, when only Chinese populations
were analyzed as one group, 29% of the total genetic
variation corresponded to differences between popula-
tions and the remaining 71% was the result of varia-
tion between individuals within populations. De Marchi
et al. (2006) used the Gst index (Nei, 1973) to estimate
the percentage of the total variation in Veneto chicken
breeds and reported a value of 0.40 for Gst across loci,
indicating that 40% of the total gene diversity was ob-
served between breeds, while the remaining 60% was ac-
counted for by the within-breed component of variation.
As expected, within-individual variation diminished to
52% when Chinese populations were grouped based
on 2 eggshell color types, while differences between
groups increased to 43%, and 5% of total variation also
1784 DALIRSEFAT ET AL.
corresponded to the between-population within-group
variation. This is apparently a result of higher genetic
similarity between each eggshell color type group, which
is in agreement with low genetic distance between pop-
ulations within these 2 groups (Table 3). When White
Leghorn chickens were included in the analysis, the pro-
portion of variation component was not influenced and
showed a very close proportion to those of the second
data set.
The number of migrants per generation (Nm) val-
ues showed relatively high influence of gene flow and
genetic drift between populations with same eggshell
color. Among populations, Lushi Blue and Jingmen
Blue showed a substantial value of gene flow, which
is consistent with the geographical locations of these 2
populations (Figure 1). However, a relatively high de-
gree of gene flow between Lindian Blue and Yimeng
Blue seems to contradict their geographical distribution
in 2 distinct provinces in China. The similarity of these
2 blue-shelled chicken populations may be explained by
the famous historical events of human mass immigra-
tion from Shandong province to the northeast of China
since 1644 in the Qing Dynasty. Genetic variation will
give rise to considerable differentiation where Nm < 1
but not where Nm > 1 (Slatkin, 1987). In the present
study, the estimated number of migrants (Nm) between
populations within each group based on eggshell color
type was significantly higher than an earlier estimate
for 15 Chinese indigenous chicken breeds using 29 mi-
crosatellite markers (Chen et al., 2008), and implies
substantial gene flow between these populations, re-
sulting in low measures of genetic differentiation and
inbreeding.
The UPGMA tree based on Reynolds’ genetic dis-
tances (Figure 2) clearly distinguished the blue-shelled
populations from the brown-shelled populations, which
further confirmed the results obtained from PCoA and
STRUCTURE-based clustering. In the UPGMA tree,
Lushi Blue and Jingmen Blue chickens clustered to-
gether and were supported by a low value of FST
and high value of gene flow (Table 3), indicating a
close genetic relationship between the 2 populations.
In a lower level of relationship, these 2 populations to-
gether with Wulong Blue and Dongxiang Blue located
in a sub-cluster. A possible explanation is that Henan
(Lushi chickens), Hubei (Jingmen chickens), Chongqing
(Wulong chickens), and Jiangxi (Dongxiang chickens)
Provinces are geographically close to each other, rais-
ing the possibility of interbreeding. The high gene flow
and low FST values between these populations (Table 3)
supported this close clustering of the populations. The 4
populations did not separate during the STRUCTURE
runs from K = 2 to 6. This close genetic association
may arise from a common genetic background or migra-
tion. Considering the geographical distribution of the
populations, one would have expected a higher level of
differentiation between the Yimeng Blue (from Shan-
dong Province) and Lindian Blue (from Heilongjiang
Province). Instead, based on results from all analyses
in this study, Yimeng Blue and Lindian Blue chick-
ens together formed a subcluster which is supported
by extremely low genetic distance (0.008) and genetic
differentiation (FST = 0.004) with a considerably high
value of gene flow (Nm = 64) between the 2 populations
(Table 3). Furthermore, there was high genetic variabil-
ity and low inbreeding coefficients (Table 2) in both
populations. This hypothesis was also confirmed by
PCoA and STRUCTURE results, as the populations
did not separate from K = 2 to 7.
As expected, Dongxiang Brown and Lushi Brown
chickens grouped in a different subcluster from all
blue-shelled chickens. These 2 populations have same
origin as Dongxiang Blue and Lushi Blue, but a
different oocyan genotype resulting to a different phe-
notype. Divergence between chicken populations with
different types of eggshell color is obviously due to the
SLCO1B3 gene controlling eggshell pigmentation. Sub-
stantial SNP dissimilarities between blue eggshell and
brown eggshell types within same population may ex-
plain why the brown-shelled chickens formed a different
cluster at lower K values.
White Leghorn chicken as an outgroup population
was expected to form a fully separate cluster from the
Chinese chickens, but it clustered with brown-shelled
chickens. STRUCTURE and PCoA results further sup-
ported this result, which may be due to more SNPs in
the SLCO1B3 gene of White Leghorn being shared with
brown-shelled rather than blue-shelled types.
In our study, the STRUCTURE analysis clustered in-
dividuals into separate populations or groups of closely
related populations, and suggested that the blue-shelled
populations are mixture populations (Figure 4). The
obvious mixed nature of these populations is consis-
tent with the high estimates of gene flow between them
(Table 3).
Aligning EAV–HP insertions of Chinese blue-shelled
chickens in this study revealed no polymorphism,
whereas Wragg et al. (2013) identified 5 polymorphisms
through aligning their EAV–HP sequence of Dongxiang
(GenBank Accession No.: KC632577) to that of Wang
et al. (2013; GenBank Accession No.: JF837512), re-
sulting in a sequence divergence of 0.1%. It is notewor-
thy that our EAV–HP sequences of 5 Chinese chick-
ens were fully identical to that of Dongxiang sequenced
by Wragg et al. (2013), but not to that sequenced by
Wang et al. (2013). In addition, different host inte-
gration sites and DNA sequences have been identified
for the Chinese chickens and Mapuche/Araucana fowls
(Wragg et al., 2013). With the knowledge of distinct ge-
nomic insertion sites, our results agree with recent stud-
ies (Wragg et al., 2013; Wang et al., 2013) and clearly
indicate the independent acquisition of the oocyan phe-
notype in native Asian and South American chickens.
As cited by Wragg et al. (2013), according to histor-
ical evidence, the oocyan phenotype has been present
since at least 500 years ago in the Dongxiang chicken in
Asia (Gao et al., 2008), and since the late 19th century
with a wide geographic distribution in South America
 PHYLOGENETIC ANALYSIS OF CHINESE CHICKENS
(Castello, 1924). The lack of divergence in the Long
terminal repeat (LTR) sequences of the EAV–HP in-
sertions within the Chinese breeds and the Mapuche
fowl supports a relatively recent integration event on
both continents (Wragg et al., 2013).
In conclusion, the present study confirms that SNP
information derived from the whole genomic region
of the SLCO1B3 gene is able to discriminate blue-
shelled from nonblue-shelled chickens, and distinguish
populations within each group. Relatively low genetic
diversity was observed in the 8 Chinese indigenous
chicken populations. The populations that contribute to
each eggshell color type are threatened by uncontrolled
breeding between them, and therefore are at risk to
become genetically uniform in the future. As Ibeagha-
Awemu and Erhardt (2005) suggested, this unfortunate
situation can be avoided by putting in place effective
breeding measures and management practices aimed at
controlling high genetic exchanges between the popula-
tions and preserving the typical/special characteristics
of each population. An initial step towards achieving
these goals might be to geographically define the breeds
while allowing for normal traditional systems of man-
agement (Ibeagha-Awemu and Erhardt, 2005).
We also found no polymorphism in EAV–HP inser-
tion sequences within Chinese blue-shelled populations,
demonstrating their failure to differentiate these pop-
ulations. However, this information might be useful to
identify variation between Chinese blue-shelled chick-
ens and Mapuche/Araucana fowls from South America
and Europe.
ACKNOWLEDGMENTS
The National 863 Project (2013AA102501) and the
Natural Science Foundation of China (31072024) are
acknowledged for financial support of this study. We
also thank breed conservation farm and the contacts
(Qigui Wang in the Poultry Institute of the Chongqing
Academy of Animal Science, Hui Li and Li Leng in
Northeast Agricultural University, Xiangtao Kang and
Guirong Sun in Henan Agricultural University, Jian-
sheng Xu in Jiangxi Donghua livestock Co., Ltd., Jun-
jiang Long in Shandong Longsheng agriculture and an-
imal husbandry Group Co., Ltd., Yan Yang in Hubei
Shendi Agriculture Science and Trade Co., Ltd.) in
the 5 provinces of China for providing samples of Wu-
long, Lindian, Lushi, Dongxiang, Yimeng, and Jing-
men chicken. Our appreciation goes to Mohsen Falahati
(University of Tehran, Iran) for his helpful technical as-
sistance during STRUCTURE analyses.
SUPPLEMENTARY DATA
Table S1. F-Statistics and estimates of Nm among
all population for each locus
1785
Table S2. Output of the Evanno method results.
Yellow highlight shows the largest value in the Delta K
column.
Table S3. Proportion of analyzed chicken popula-
tions in each cluster (K = 7).
Figure S1. Plot of mean likelihood L(K) and vari-
ance per K value from STRUCTURE on a dataset con-
taining 318 individuals genotyped for 21 polymorphic
SNP loci.
Figure S2. Evanno et al. (2005) plots for detecting
the number of K groups that best fit the data
Figure S3. Partial schematic diagram of LTR se-
quence alignment in six Chinese blue eggshell and Ma-
puche/Araucana chickens. Forward strand multiple se-
quence alignment of the host (Gallus gallus OATP1B3)
sequence upstream (a) and downstream (b) of each in-
tegration site is highlighted in green, with the LTR se-
quence of the EAV–HP highlighted in yellow, and the
target site duplication highlighted in red.
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