Phylogeny of 19 Indigenous Sheep Populations in Northwestern China Inferred from Mitochondrial DNA Control Region
To study genetic information of the local sheep populations of Northwestern China and their phylogenetic positions in relation to other Asian populations and European sheep, the genetic diversity and phylogeny evolution of 223 individuals from 19 indigenous sheep populations in Northwestern China and one Western breed, Texel were analyzed with entire sequences of mitochondria DNA control region. The length of the sequences was considerably variable between 1103 and 1256 bp. Phylogenetic analysis indicated that 3 mtDNA lineages A, B and C were found in all sampled populations, except for the absence of lineage C in two populations. Phylogenetic tree and median joining network were constructed with the sequenced mtDNA control region and both the phylogenetic tree and network demonstrated that there were three independent clusters and some sequences deviated from cluster A and C. Neighbor-joining tree constructed based on genetic distances showed a multi-maternal originality of the nineteen Xinjiang local sheep populations, which were clustered with the sheep groups in other parts of China, Central Asia, Turkey and India. The high genetic diversity of Xinjiang local sheep populations suggests that they may have distinct maternal origins and more complicated phylogenetic history than the other Chinese sheep breeds.
Received: March 28, 2010;
Accepted: May 06, 2010;
Published: July 10, 2010
Mitochondrial DNA (mtDNA) is an important material for phylogenetic inference
and has been widely used to explore genetic diversities among various groups
of organisms. MtDNA data from wild and domestic sheep revealed that there were
no contributions from urial and argali species to domestic sheep (Hindleder
et al., 1998, 2002) and 3 maternal lineages
A, B and C were identified in modern domestic sheep breeds sampled from different
geographical regions of the world (Hiendleder et al.,
1998, 2002; Meadows et al.,
2007; Wood and Phua, 1996; Guo
et al., 2005; Pedrosa et al., 2005; Tsunada
et al., 2010).
Xinjiang Uyghur Autonomous Region in the northwestern China spanning over 1.6
million km2, which constitutes one-sixth of China's total territory,
as a hub of Silk Road, playing an important role to linking Middle East and
Europe and still preserves a multi-ethnic, multi-linguistic and multi-religious
features up to now. The area is a melting pot for not only different cultures,
but also livestock breeds. The Uyghur, Kazakh, Kyrgyz, Mongolian, Tajik and
the other ethnic groups in this region all have long nomadic history and they
raised and preserved their livestock breeds under different cultures, geographical
isolation and self-sufficient production systems. Up to now, Xinjiang still
has a large number of indigenous sheep populations.
The numbers of sheep breeds in Xinjiang are different in different sources
(ACS, 1964; Yu et al., 1992;
Tu et al., 1989; Chen and Xu,
2004). Some sheep breeds still have not been registered officially due to
the lack of genetic information and study. Genetic information about local sheep
breeds of Xinjiang and their varieties and its phylogenetic position in relation
to neighbor Central Asian Republics and the other areas of China have not been
The aim of this research is to study the genetic diversity and the phylogeny
of indigenous sheep populations domesticated in Xinjiang. In the present study,
we compared our data with the available sheep sequences in Genbank database
(Guo et al., 2005; Hiendleder,
1998; Hiendleder et al., 1998,
2002; Pedrosa et al., 2005) and solid evidences
achieved from the mtDNA analysis showed that the indigenous sheep populations
in Xinjiang have multi-maternal origins.
MATERIALS AND METHODS
Sample collection and genomic DNA extraction: This research project
was conducted from October 2008 to December 2009 in Beijing, including sampling
from Xinjiang local sheep from June 2008 to October 2008 and January 2009 to
Ear tissues of nineteen sheep populations were collected from small and remote
villages in Xinjiang and blood samples of Texel, a European breed, were collected
from a farm in Beijing. Samples were randomly collected from each population
and the geographical distribution and the pedigree information provided by the
owners and local farmers were also taken into consideration. Ear tissues were
collected and stored at -20°C in 75% ethanol before DNA extraction. Total
Genomic DNA was extracted from ear tissue and blood by a standard phenol-chloroform
extraction method with minor modification (Sambrook et
al., 1989). Geographical distribution of the sampled populations was
shown in Fig. 1.
DNA amplification and sequencing: To select individuals from each population
for mtDNA sequencing, the two pairs of primers and SSCP analysis described previously
were used (Guo et al., 2005). The PCR amplification
for SSCP analysis was carried out in a 25 μL reaction mixture containing
20-100 ng DNA template, 1.0 μM of each primer, 200 μmol of dNTPs,
1x PCR buffer (including 1.5 mM MgCl2 and one unit of Taq DNA polymerase
(Huitian Dongfang Co., Beijing, China). The PCR conditions consisted of an initial
denaturation at 94°C for 5 min, followed by 33 cycles at 94°C for 30
sec, 58°C for 30 sec, 72°C for 45 sec and a final extension of 10 min
at 72°C. The PCR Amplification was performed in an Effondorf Master Gradient
Programmable Thermal Controller (Effondorf Inc., Germany).
The SSCP vertical electrophoresis was performed at 10V cm-1 in 7
min at first and following 5V cm-1 at RT (room temperature) for 14
h, using the DYY-6C power system (Beijing 61 Factory, Beijing, China). The gel
was silver stained and different lineages visually determined and scored (Guo
et al., 2005). The DNA Samples for sequencing entire mitochondrial
control region were randomly selected from each lineage and amplified with the
primers described previously (Hiendleder et al.,
2002). The purified products were bidirectly sequenced using BigDye
Terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) on
ABI PRISM 3700 DNA Analyzer. Two following internal primers were used for sequencing
in present study.
SGM-C378 (5-ATGCGTATCCTGTCCATTAGA-3) and SGM-C379 (5-TGAAGAAAGAAC
Data analysis: Data analyzed in the present study comprised the sequences
of entire mtDNA control region of 223 from the nineteen sheep populations in
Xinjiang and one western breed, Texel and other 59 known sheep sequences obtained
from Genbank, including the breeds in China, Turkey, Tajikistan, Kazakhstan,
India and wild sheep like Ovis ammon nigrimontana, Ovis vignei bochariensis,
Ovis ammon (argali). The breed names of sequences retrieved from Genbank, their
location and Genbank access number are shown in Table 1. All
sequences were manually edited using the Bioedit package (Hall,
1999) and aligned by using the Clustal W program (Thompson
et al., 1994).
|| Sequences obtained from Genbank
Phylogenetic and molecular evolutionary analyses were conducted using MEGA
version 4 (Tamura et al., 2007), with a Kimura
2-parameter (transition only) model and a bootstrap (1000 replications) test.
A pairwise distance matrix between mtDNA haplotypes was calculated and a Neighbor
Joining (NJ) tree was constructed based on the nucleotide p-distances and other
parameters were set at the default values.
The Median Joining (MJ) networks (Bandelt et al.,
1999) were drawn using the program Network 184.108.40.206 (http://www.fluxus-engineering.com)
to investigate the possible relationships among haplotypes. Nucleotide and haplotype
diversities were computed using DnaSP 5.10.00 (Librado and
Rozas, 2009). Fus Fs statistics (Fu, 1997)
and Mismatch distribution, were computed using Arlequin3.11 (http://www.cmpg.unibe.ch/software/arlequin3/).
RESULTS AND DISCUSSION
Two hundred and twenty three mtDNA samples from the nineteen local breeds in
Xinjiang and one Western breed were sequenced. The length of the sequences in
Xinjiang sheep was considerably variable between 1103 and 1256 bp. The sample
information and number of haplotypes, haplotypes diversity, observed lineage
and nucleotide diversity of each population were shown in Table
We constructed a Neighbor-joining phylogenetic tree (Saitou
and Nei, 1987) of the 19 Xinjiang local populations with Hu, Tong, Han,
Mongolian, Tibetan, Kivircik, Daglic, Akkaraman, Gizarr, Edilbey, Astrachan,
Bonpala, Garole, Ovis ammon nigrimontana, Ovis vignei bochariensis and Ovis
ammon argali which retrieved from Genbank. The results of the phylogenetic analysis
showed the same three clades, which were reconciled with haplotypes A, B and
C. The NJ tree constructed by Mega 4.0 was shown in Fig. 2.
|| The sample information and some diversity indices of the
|SE: Standard error
||Phylogenetic relationships of Xinjiang local sheep breeds.
Sequences of Hu, Tong, Han, Mongolian, Tibetan, Kivircik, Daglic, Akkaraman,
Gizarr, Edilbey, Astrachan, Bonpala, Garole, Ovis ammon nigrimontana, Ovis
vignei bochariensis and Ovis ammon argali were retrieved from Genbank
All of the sheep populations in Xinjiang were clustered with other sheep breeds
of China, such as Mongolian Sheep, Tibetan Sheep and also the breeds in other
countries mainly around Silk Road, such as Kivircik, Daglic, Akkaraman, Gizarr,
Edilbey, Astrachan, Bonpala and Garole, which correspond with their geographical
distribution. But surprisingly, the Tarim population was not clustered with
the sheep breeds in Xinjiang, but with Gizarr, Astrachan, Bonpala and Garole;The
Emil population was clustered with Tibetan Sheep at first and then clustered
with Edilbey, Daglic, Kivircik, Daglic and Akkaraman, despite of long geographical
distances between them. Fus Fs statistics (Fu, 1997)
gives a very significant negative value when a population expansion occurs.
Based on the results of the Fus Fs statistic and mismatch distributions
(data not shown) from Arlequin, the probability of observing a random neutral
sample with a number of alleles is similar than the observed value. The Fs values
for haplotypes A, B and C were -76.282 (p<0.000), -22.510 (p<0.000) and
-9.334 (p<0.00600), respectively, which suggested that lineages A, B and
C depart significantly from the neutral model and have a quite different demographical
history. A network analysis was performed to define the genetic structure between
the lineages. The results showed that the 3 clusters correspond with the phylogenetic
clades (Fig. 3).
||Network of three lineages in nineteen sheep populations in
Xinjiang. The hollow circles are 223 samples and the solid ones indicate
|| Parameters of mtDNA variability within the three clusters
in Xinjiang local sheep
Each clade was clearly isolated, but there was some deviation in clade A and
clade C, showing more than one common maternal ancestor in the two clades. Parameters
of mtDNA variability within each cluster were given in Table 3.
To study the origins of Xinjiang indigenous sheep populations, 223 entire D-loop
regions of different Xinjiang sheep populations were sequenced and a phylogeny
tree was constructed with the sequence data. We conducted a comprehensive phylogenetic
analysis of the nineteen Xinjiang local sheep breeds to obtain information about
the genetic origins and evolutionary history compared with other sheep breeds/populations
of different regions of China and other countries.
The length of the sequences varies between 1103 and 1256 bp. The difference
of length of the sequences was caused by 3-5 copies of a 75 bp tandem repeated
sequence. In earlier studies, three maternal lineages A (Asian type), B (European
type) and C (Central Asian and Middle East type) were identified in modern domestic
sheep breeds sampled from different geographical regions of the world (Hiendleder
et al., 1998, 2002; Meadows
et al., 2007; Wood and Phua, 1996; Guo
et al., 2005; Pedrosa et al., 2005; Tsunada
et al., 2010; Chang, 2009) and a small number
of individuals of maternal lineage D (Caucasian type) also were reported before.
In the present study, all of lineage A, B and C were found in our studied populations.
There were some studies on phylogenetic analysis carried out about the local
sheep breeds of Xinjiang (Wang et al., 2007;
Yang and Zhao, 2002; Tang et
al., 2009; Tsunada et al., 2010; Guo
et al., 2005) and the results of them suggested that Xinjiang sheep
populations are mainly maternally originated from and influenced by Kazakh and
Mongolian sheep. However, there were few studies about the origins and phylogeny
of the many unregistered Xinjiang sheep populations. Sheep breeds in China are
mainly categorized into Mongolian, Tibetan and Kazakh and Yunnan sheep groups
based on archaeological, morphological, historical and available molecular genetic
information (Chang, 2009). Previous studies showed that
some Xinjiang local sheep populations share the same haplotypes with other sheep
breeds of China and local sheep populations in Xinjiang belong to Kazakh sheep
groups and have close genetic relationship with Mongolian Sheep (Yu
et al., 1992; Tu et al., 1989; Chen
and Xu, 2004; Chang, 2009), but in present study,
there are some exceptional cases. It is very interesting that the Tarim population
was not clustered with other sheep populations or breeds in China, but with
Gizarr, Astrachan, Bonpala and Garole; The Emil population was clustered with
Tibetan at first and then clustered with Edilbey, Daglic, Kivircik, Daglic and
Akkaraman, despite the thousands kilometer distance between them. Some individuals
are deviated from clade A and C. Unlike lineage B(European type), lineage A
and C had more complex phylogenetic branches, which indicates the two lineages
might be derived from a number of founders instead of a single common ancestor.
Xinjiang local sheep breeds may have more complicated phylogenetic history than
other Chinese breeds. The populations like Tarim, Yengsar, Barchuq and Lopnor
shows less genetic distance from Kazakh and Mongolian sheep breeds and were
clustered with Tajikistan and Indian sheeps.
Most of Xinjiang sheep populations were closed to Asian types. However, one
population showed genetic links with the Turkish and Indian sheep breeds. The
samples used in the study were mostly distributed in remote and undeveloped
areas and hardly influenced by foreign sheep breeds, so they could represent
the genetic situations of these domestic sheep populations. But due to the limited
sequencing number of each population, it could not be confirmed whether the
populations had migrated from Turkish and Indian sheep breeds or Turkish and
Indian sheep breeds originated from Tarim.
In the present study, some sheep populations from the same region were clustered
into different subgroups, like Tarim, Lopnor and Kucha which were from South
Xinjiang, but our results showed that they belonged to different subgroups,
respectively. On the other hand, some populations from different areas were
clustered to the same subgroups which suggested that there might be some gene
flows between sheep populations. Although, this region was separated by mountains,
large deserts, relatively isolated oasis and long geographical distances, gene
flows probably existed between the different populations in these regions along
with Silk Road.
Historical and archaeological events indicated that Europoid people lived in
Xinjiang from pre-historic times. In ancient time, Xinjiang territory was usually
divided by many local oasis rulers like Huns, Turkic and Mongoloid or warring
empires 'chasing grass and water' (Millward, 2007). Xinjiang
were sweep away by imperators ruling over pastoralist and agrarian areas from
Iran to China. Parts of Xinjiang were often ruled together with lands which
now are now the Central Asian Republics. Later the dissolution of Turk empire
because of wars, diseases and natural disaster led to one of history's great
movement of Turkic-speaking tribes into Xinjiang and across Central Eurasia.
As a result of the movement, there are now several Turkic language-spoken ethnic
groups in Xinjiang including Turkish, Uyghur, Uzbek, Kazakh, Kyrgyz and others.
The movement also led to migration and integrations of livestock populations.
For thousands of years, there were active commodities exchanges along the famous
Silk Road. Sheep, as a main livestock in these regions, might also be under
the exchange. The above information and historical events may provide some clues
for us to understand why the two Xinjiang local sheep populations were genetically
closed to Turkish and Indian sheep breeds.
Present results show that genetic relationships of the two populations with
Mongolian, Tibetan and Kazakh breeds are not close and the origin and migration
history of Tarim and Hotan populations in South Xinjiang were independent from
the other populations in the same areas. The high genetic diversity of Xinjiang
local sheep populations implies that they may have distinctive origins. According
to NJ phylogenetic tree and genetic distances, Xinjiang local sheep populations
may be classified into four subgroups: (1) Altay, Turpan,Tarim, Yengsar, Barchuq
and Lopnor. (2) Emil, Chira, Barikol, Bashbay, Tashqurghan, Kyrgyz, Nowesh,
Bayinbulaq and Kazakh. (3) Yecheng, Kucha and Hotan. (4) Dolan. The first and
third subgroups might be derived from a number of founders instead of a common
This study provided molecular evidences of Xinjiang local sheep of genetic
diversities, which showed that Xinjiang local sheep populations may have multiple
maternal origins, which are not only related to Kazak, Mongolian and Tibetan
origin, but also to other two ancestors of Hotan and Tarim.
The results would be also helpful for us to understand the diversity and origins
of Central Asian and Middle East domestic sheep. Whether Hotan and Tarim are
derived from two separate maternal origins needs to enunciate with more evidences
from the further studies on phylogeny of sheep populations in Afghanistan, Pakistan,
Iran and Central Asian Republics.
The research was supported by the National Basic Research Program of China
(973 Project) (code: 2006CB102100).
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