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Copyright ῌ ,*+*, Japan Poultry Science Association.

Bing Zhou+, Shi-Yi Chen+, ,, Qing Zhu+, Yong-Gang Yao, and Yi-Ping Liu+
College of Animal Science and Technology, Sichuan Agricultural University, Ya’an, Sichuan, 0,/*+., China
Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences &
Yunnan Province, Kunming Institute of Zoology, Kunming, Yunnan, 0/*,,-, China

Silkie is a famous black-bone chicken breed with beautiful silky feather. The unique medical property of this chicken was
recorded in Chinese traditional medicine dictionary about 1** years ago. In this study, we analyzed the mtDNA D-loop
sequence variation of ,0 Bairong Silkies from Fujian Province, China, together with +** reported Silkie mtDNAs from China
and Japan, and studied their matrilineal components and genetic relationship. A total of ,+ haplotypes were detected, which
could be assigned to six haplogroups (A-E, G). Among them, haplogroups D and G were exclusively presented in Japanese
Silkies and Chinese Silkies, respectively. Chinese Silkies had higher frequency of lineages belonging to haplogroups A, B, and
E, and lower frequency of haplogroup C than Japanese Silkies. For the four Chinese Silkie populations, most of samples of
Taihe, Chengdu, and Hubei Silkies were grouped in haplogroups A, B, and C, whereas most of Bairong Silkies were grouped
in haplogroup E. Five haplotypes were shared by Japanese and Chinese Silkies. The genetic diversity of each Silkie population
varied, but the overall diversity of Chinese Silkies was similar to that of Japanese Silkies. Taken together, our results
confirmed the genetic connection between Chinese and Japanese Silkies, but also clearly showed that the matrilineal genetic
structures of Chinese and Japanese Silkies had some di#erences.

Key words: d-loop, genetic diversity, matrilineal origin, mtDNA, silkies

J. Poult. Sci., .1: ,,ῌ,1, ,*+*

Although most of domesticated chickens are used for

meat consumption and egg production, the ornamental
The modern domestic chickens are now distributed types have been prevailed worldwide today. Among them,
worldwide and have evolved along with the development the Silkie (Gallus gallus domesticus) is well known for its
of human civilization. The origin and phylogeographic unique flu#y plumage feeling like silk. In China, Silkie is
history of chickens across Eurasia have been extensively specially characterized by its unique medical property,
elucidated based on mitochondrial DNA (mtDNA) se- which was recorded in Chinese traditional medicine dic-
quence variation (Fumihito et al., +33., +330; Liu et al., tionary about 1** years ago (Xie, +33/). There is no clear
,**0a). The wildly accepted concept is that the progeni- distinction between the Silkies and other chicken breeds at
tor of domestic chickens is red jungle fowls (Gallus the matrilineal component level (Fu et al., ,**,; Liu et al.,
gallus), and the highly divergent mtDNA haplogroups in ,**0a). The exact scenario regarding where or when
domestic populations would suggest for multiple domes- Silkies with the fur-like plumage were first cultivated was
tications in di#erent regions, such as Yunnan, South and unknown. Most of the existing conventional literatures
Southwest China and/or surrounding areas, as well as the suggested for an origin in China. Other places in South-
Indian subcontinent (Liu et al., ,**0a). As the initially east Asia, such as India, have also been proposed as the
domesticated chickens were adapted to new environments cultivation center for the Silkie breeds (Arisawa et al.,
and were subjected to di#erent artificial selection pres- ,**0). The domestication history of chicken in China
sures, populations with new characteristics were de- could be dated back to /.** BC (West and Zhou, +323),
veloped and further formed local breeds (Zárate et al., but the first detailed description for the Silkies was
,**0). recorded only 1** years ago (Xie, +33/). Nowadays,
Received: June ., ,**3, Accepted: October /, ,**3
there are eight Chinese Silkie breeds/populations regis-
Released Online Advance Publication: December +*, ,**3 tered in the Domestic Animal Diversity Information
Correspondence: Prof. Y. Liu, College of Animal Science and Tech- System (DAD-IS, ,**1) of the Food and Agriculture
nology, Sichuan Agricultural University, Ya’an, Sichuan province, Organization of the United Nations (
0,/*+., China. (E-mail: liuyp/ All these breeds/populations could be assorted
Zhou et al.: Matrilineal Components of Silkies 23

into white feather type and black feather type based on the described in our previous study (Liu et al., ,**0a) and
feather color (Qiu et al., +322). Among them, the Taihe were deposited in GenBank under accession numbers GQ
Silkies and Bairong Silkies, which are distributed in ,,133--GQ,,2*+2.
Jiangxi Province and Fujian Province, respectively, are The previously reported sequences (,1 Taihe Silkies
thought to be the ancestor populations of modern Silkies from Jiangxi Province, +/ Chengdu Silkies from Sichuan
in China. Subsequently, these two breeds were di#used Province, ,. Hubei Silkies from Hubei Province, China;
into other regions (Qiu et al., +322; Xie, +33/). Unfortu- Liu et al., ,**0a) were also used for comparison in this
nately, there is no strong evidence, especially for molecu- study. Thirty-four Japanese Silkies were retrieved from
lar data, to support this hypothesis. GenBank. All together, +,0 mtDNA D-loop partial se-
Silkie is one of the most popular and ubiquitous orna- quences were analyzed in this study (Table +).
mental breeds in Japan. Silkie is called “Ukokkei” in Data Analysis
Japanese and is mainly distributed in Tokyo, Mie, Osaka, The mtDNA sequences were aligned with the reference
Hiroshima, Yamaguchi and Kagawa Prefectures (Chang sequence of G. g. domesticus mitochondrial genome
and Huang, ,**-). There are about /* native chicken (Desjardins and Morais, +33*). Sequence variations were
breeds in Japan, which could be divided into entertain- exported using MEGA -.+ (Kumar et al., ,**.). We fol-
ment, egg, and/or meat production groups. The enter- lowed the classification system in our previous report (Liu
tainment group could be further classified into two sub- et al., ,**0a) to classify each mtDNA into respective
groups according to their introduction history, with one haplogroups/clades. An unrooted Neighbor-joining (NJ)
being introduced to Japan more than ,, *** years ago and tree of the haplotypes was subsequently constructed using
the other one during the early Edo Era (+0*-ῌ+201) from the Kimura two-parameter model in MEGA -.+, follow-
China or India (Chang and Huang, ,**-; Arisawa et al., ing the same rationale as discussed in our previous study
,**0). Based on these founder breeds, other Japanese (Liu et al., ,**0a). To provide a better view of the genetic
breeds were subsequently developed via artificial selection relationship of the haplotypes in these geographic popula-
programs by the end of the Edo Era (Arisawa et al., tions of Silkies, a median-joining network was constructed
,**0). Komiyama and colleagues (,**-, ,**.) suggested using program Network -.+ (http://www.fluxus-
that all Japanese domesticated chickens were originated; Bandelt et al., +333). Fi-
independently from Southeast Asia and the mainland nally, the haplotype diversity (h) and nucleotide diversity
China. Our later reanalysis of the gamecock mtDNAs in (p) for each Silkie population (Nei, +321) were estimated
China and Japan demonstrated a genetic pattern consis- using DnaSP ..+* (Rozas et al., ,**-).
tent with the proposed dual origin of Japanese gamecocks,
but also left room for a single origin of Japanese game-
cocks from China (Liu et al., ,**0b). Recently, Oka et al. A total of ,1 polymorphic sites, including ,0 transitions
(,**1) provided further mtDNA data to support the and one transversion, were found in +,0 mtDNA se-
multiple origins of Japanese native chickens. quences (each with a length of /+3 bp). Twenty-one hap-
Mitochondrial DNA has been proved to be a very useful lotypes were defined and all of them could be found in the
genetic marker in elucidating the origin of domestic data set reported by Liu et al. (,**0a) (Fig. +). The ,+
animals, such as duck (He et al., ,**2), Cattle (Lai et al., haplotypes could be classified into six haplogroups (A-E,
,**0; Chen et al., ,**2), and Goat (Liu et al., ,**3). The G). Briefly, six haplotypes belonged to haplogroup A, two
domestication history and phylogeographical relationship haplotypes in haplogroup B, two haplotypes in haplogroup
of modern chickens in China and Japan have been C, three haplotypes in D haplogroup, seven haplotypes in
elaborately explored by using mtDNA sequence variations haplogroup E, and one haplotype in haplogroup G.
(Fumihito et al., +33., +330; Komiyama et al., ,**-, Among them, haplotypes A,, C+, B+, E+, and B+- were
,**.; Liu et al., ,**0a, b; Oka et al., ,**1), as well as, dominant and were shared by ,-, ,,, ,+, ,*, and +,
microsatellite DNA polymorphisms (Takahashi et al., samples, respectively. Nine haplotypes occurred in one
+332). However, the matrilineal components of the Silkie sample. Each of the seven haplotypes (A0, A++, A+2, D0,
breeds distributed in China and Japan and their genetic D+-, E0, and E3) occurred in ,ῌ. samples. Haplotypes A
relationship have not been su$ciently characterized or , and B+ were shared by individuals from all four Chinese
discussed. In this study, we compared the mtDNA con- Silkie populations and one Japanese Silkie population.
trol region (D-loop) partial sequence variation of the However, haplotypes E+ was shared by +3 individuals
Silkies from China and Japan. Our results provided in- exclusively from the Bairong Silkies and one Japanese
sightful information regarding the breed formation histo- Silkie. When we counted the number of haplotypes ac-
ry and subsequent di#usion route of the Silkie. cording to the Silkie populations, +- haplotypes were
detected in Japanese Silkies, five haplotypes in Bairong
Materials and Methods
Silkies, seven haplotypes in Taihe Silkies, five haplotypes
Sampling and Reported Data in Chengdu Silkies, and five haplotypes in Hubei Silkies.
We collected ,0 Bairong Silkies from Fujian Province. The phylogenetic relationship among the six haplo-
These samples were sequenced according to the procedure groups was displayed in the NJ tree (Fig. ,a), and was
24 J. Poult. Sci., .1 (+)

Table +. Sample information and genetic diversity of Silkies

Haplotype Nucleotide
Breed No. Location Accession Reference
diversity diversity
Bairong Silkie ,0 Fujian, China GQ,,133--GQ,,2*+2 *4.0,῏*4++. *4**/*3῏*4**+2, This study
Taihe Silkie ,1 Jiangxi, China AF/+,,,+-AF/+,,--, *42-2῏*4*-. *4*++,-῏*4**+*3 Liu et al., ,**0a
Chengdu Silkie +/ Sichuan, China *42.2῏*4*0+ *4**2++῏*4**,*, Liu et al., ,**0a
Hubei Silkie ,. Hubei, China *410.῏*4*., *4**2+,῏*4**+.0 Liu et al., ,**0a
Subtotal in
3, ῎ ῎ *42/+῏*4*+/ *4*++02῏*4***/,
AB**11--, AB**11-/,
AB**11.*, AB**11.0, Data
AB**11.1; deposited in
Japanese Silkie -. Japan *42.+῏*4*/0 *4*+,+3῏*4**+*0
AB++.*1*, AB++.*1+; GenBank by
AB,0-3.1-AB,0-31*, Miyake, T.
Total +,0 ῎ ῎ *422,῏*4*+, *4*+,32῏*4***-*

Fig. +. mtDNA sequence variations of ,+ haplotypes

identified in +,0 Silkie samples collected in this study
and from published sources. The haplotypes were aligned
to G. g. domesticus complete mtDNA sequence (Desjardins
and Morais, +33*) and were defined according the
classification system in our previous study (Liu et al.,
,**0a). The number of individuals sharing the same
haplotype in di#erent populations is listed below the
abbreviations for each population (BR-Bairong Silkie;
TH-Taihe Silkie; CD-Chengdu Silkie; HB-Hubei Silkie;
JP-Japanese Silkie). The column “T” shows the total
number of individuals shared each haplotype. Dots (ῌ)
denote the identity with the reference sequence. Short
lines (ῌ) represent the absence of certain haplotype in the

further demonstrated by a network graph (Fig. ,b). The two samples belonging to haplogroups E and G, respec-
topology of the tree based on the Silkie haplotypes was tively. In contrast, ,, Bairong Silkies (2..0ῌ) from
consistent with the pattern observed in a large domestic Fujian, China, were distributed in haplogroup E, while
chicken data set (Liu et al., ,**0a). Among the -. four samples in haplogroups A, B, and C. Among the six
Japanese Silkies, +- samples were distributed in haplo- haplogroups, D and G were exclusively found in Japanese
groups D and E, whereas the remaining ,+ samples could Silkies and Chinese Silkies, respectively. The frequencies
be classified into haplogroups A, B, and C. For the Taihe, of haplogroups A, B, and E were higher in Chinese Silkies
Chengdu, and Hubei Silkies, nearly all samples were dis- than in Japanese Silkies, but the frequency of haplogroup
tributed in haplogroups A, B, and C, with the exception of C was higher in Japanese Silkies (Table ,). Furthermore,
Zhou et al.: Matrilineal Components of Silkies 25

Fig. ,. The unrooted neighbor-joining (NJ) tree (a) and the net-
work graph (b) of the mtDNA haplotypes in Silkie samples. The
links are labeled by the nucleotide positions to designate tran-
sitions. Circle areas are proportional to haplotype frequencies.
White and black colors were denoted for Chinese and Japanese
Silkies, respectively.

Table ,. Haplogroup distribution frequency in Chinese and Japanese Silkies

Haplogroup A B C D E G
-+4/ῌ -,40ῌ 342ῌ ,/4*ῌ +4+ῌ
Chinese Silkies *
,3ῌ 3, -*ῌ 3,  3ῌ 3, ,-ῌ 3,  +ῌ 3,
++42ῌ 242ῌ .+4,ῌ ,*40ῌ +140ῌ
Japanese Silkies *
 .ῌ -.  -ῌ -. +.ῌ -.  1ῌ -.  0ῌ -.
,04,ῌ ,04,ῌ +24-ῌ /40ῌ ,-4*ῌ *42ῌ
--ῌ+,0 --ῌ+,0 ,-ῌ+,0  1ῌ+,0 ,3ῌ+,0  +ῌ+,0
Note: Haplogroups were defined in Liu et al. (,**0a).

five haplotypes were shared by Japanese Silkies and Chi- develop special and favorable strain/breed of farm ani-
nese Silkies, whereas eight haplotypes were exclusively mals has received more attention in recent years. Due to
found in Japanese Silkies. the medical property and beautiful appearance, many
The mean haplotype diversity and nucleotide diversity researchers have attempted to improve the production
were *.22,*.*+, and *.*+,32*.***-* for all chicken levels of Silkies (He, ,**-). To know the genetic structure
samples, respectively. For di#erent Silkie populations, and diversity of certain domestic animal is essential for
both the Chengdu Silkies (*.2.2*.*0+) and Japanese developing rational breeding programs. mtDNA sequence
Silkies (*.2.+*.*/0) presented a high value of haplotype variations have been proved to be a useful tool in analyz-
diversity. The Bairong Silkies had the lowest haplotype ing the genetic structure and tracing the matrilineal origin
diversity (*..0,*.++.) and nucleotide diversity (*.**/*3 of domestic birds (Liu et al., ,**0a; He et al., ,**2). Fu
*.**+2,). Overall, Chinese Silkies had a relatively equal and colleagues (,**,) studied the genetic relationship
diversity with Japanese Silkies (Table +). between Silkies and other chicken breeds using mtDNA
sequence variations, and detected no obvious di#erentia-
tion. This result was further confirmed in our subsequent
With the improvement of living standard in China, to analysis of chicken mtDNA sequences from various
26 J. Poult. Sci., .1 (+)

breeds (Liu et al., ,**0a). However, both studies did not high frequency of haplogroup D, which has been found to
discuss the genetic relationship between the Chinese and be closely related to the distribution of the pastime of cock
Japanese Silkies. fighting (Liu et al., ,**0a, b), in Japanese Silkies distin-
By employing the recently described chicken mtDNA guished itself clearly from the Chinese Silkies. In short,
classification system (Liu et al., ,**0a) and available our results provided evidence for the genetic connection
mtDNA data, we compared the matrilineal components of between Chinese and Japanese Silkies, but also
Chinese and Japanese Silkies, with an intention to learn demonstrated the di#erence between the matrilineal genet-
more about the past history of the Silkies. In particular, ic structures of these Silkies.
we collected new samples from the Bairong Silkie, which
has been thought to be one of the ancestor populations of
modern Silkies in China (Qiu et al., +322). Out of the nine The authors thank the anonymous reviewer for helpful
highly divergent mtDNA haplogroups that were defined comments on the early version of the manuscript. This
by Liu et al. (,**0a), six were found in the +,0 samples work was financially supported by the earmarked fund for
from Japanese and Chinese Silkies. Such a pattern further Modern Agro-industry Technology Research System, the
confirmed the complexity of domestic chicken matrilineal Science Fund for Young Scholars in Sichuan Province
components at the breed level. Among the four Chinese (Grant No: ZQ *,0ῌ*+1), and the National 20- Project of
Silkie populations, Bairong Silkies di#ered from the other China (No. ,**2AA+*+**+).
three breeds/populations (Taihe, Chengdu, and Hubei
Silkie) by harboring di#erent frequencies of haplogroups.
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Zhou et al.: Matrilineal Components of Silkies 27

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