遗 传 学 报 Acta Genetica Sinica, December 2006, 33 (12)：1087–1095 ISSN 0379-4172

Study on Tandem Repeat Sequence Variation in Sheep

mtDNA D-loop Region
LI Xiang-Long1,①, GONG Yuan-Fang2, LIU Zheng-Zhu2, ZHENG Gui-Ru1, ZHOU Rong-Yan1,
JIN Xiao-Min2, LI Lan-Hui1, WANG Hai-Liang1
1. College of Animal Science & Technology, Agricultural University of Hebei, Baoding 071001, China;
2. Department of Animal Science, Hebei Normal University of Science & Technology, Changli 066600, China

Abstract: The 75-nt-long tandem repeat sequence in the control region of mtDNA of 77 individuals, of which 69 were from
different indigenous sheep breeds in China and 8 were from imported breeds, was sequenced and analyzed to investigate the origin
and differentiation of Chinese indigenous sheep breeds and also the genetic diversities and relationships among them. A total of 28
variable sites were detected within 309 repeated sequences, among which 7 sites were singleton variable sites with two variants, 1
site was a singleton variable site with three variants, and 20 sites were parsimony informative sites with two variants. A total of 63
haplotypes were sorted from 28 polymorphic sites, among which two main and basic haplotypes, namely, Hap 1 and Hap 3 were
present at a much higher proportion, at 12.94% and 30.42%, respectively. It could be inferred that Chinese indigenous sheep breeds
originated from two maternal ancestors because of the maternal inheritance characteristics of the mtDNA. Altay sheep and
Kazakstan sheep are closely related and do not differentiate significantly. Mongolian sheep and Ujumuqin sheep also share a close
relationship. Tibetan sheep, Mongolian sheep, and Ujumuqin sheep have lower genetic diversity than Altay sheep and Kazakstan
sheep.
Key words: sheep; mtDNA D-loop; tandem repeat sequence; variation

Mitochondrial DNA (mtDNA) has been proven
to be useful markers to study the maternal origin of
domestic animals because of its characteristic mater-
nal inheritance. Hiendleder et al. [1,2] obtained the
complete sequence of sheep mtDNA and proved that
domestic sheep were derived from two different an-
cestral maternal sources. The polymorphism of
mtDNA on some Chinese indigenous sheep breeds
has been reported and it provided useful data for
studying the origin and differentiation of Chinese
indigenous sheep breeds [3 - 9]
. Hiendleder et al.
pointed out that the length of sheep complete se-
quence is not absolute because of the heteroplasmy
caused by the occurrence of different numbers of a
75-nt-long tandem repeat in the control region. In the
present study, the mtDNA tandem repeat sequence
was studied in 69 individuals from different indige-
nous sheep breeds in China and 8 samples from im-
ported breeds to investigate the origin and differentia-
tion of Chinese indigenous sheep breeds and also the
genetic diversities and relationships among them.
1 Materials and Methods
1. 1 Population samples and DNA extraction
Seventy-seven samples were taken from differ-
[1] ent sheep breeds: 8 from Mongolian sheep, 11 from
Ujumuqin sheep, 5 from Dorset sheep and 3 from
Merino sheep in Inner Mongolian Autonomous Re-
gion, 6 from Kazakstan sheep and 7 from Altay sheep
in Xinjiang Uigur Autonomous Region, 10 from Tan
sheep in Ningxia Hui Autonomous Region, 10 from

Received: 2006-03-23; Accepted: 2006-05-01

This work was supported by Natural Science Foundation of Hebei Province (No. 302423).
① Corresponding author. E-mail: lixianglongcn@yahoo.com
 1088
Tibetan sheep in Gansu and Sichuan Provinces, 10
from Hu sheep in Jiangsu Province, and 7 from Small
Tail Han sheep in Shandong Province. All were kid-
ney samples preserved in 75% alcohol. In all cases,
samples were taken from different villages and farms,
and owners were questioned in detail to ensure that
the samples were not related. Genomic DNA was
extracted using the Wizard Genomic DNA Extraction
Kit (Promega, China) according to manufacturer’s
instructions.
1. 2 Primer design and PCR amplification
Primer 3 (http://www.genome.wi.mit.edu/cgibin/
primer/primer3_www.cgi) was used to design primers
for amplifying of part of the D-loop region according
to sheep mtDNA sequence (AF039578). D-loop re-
gion with a length of 1 055 bp would be amplified by
forward (5′-AACTCCCAAACATACAACACGG-3′)
and reverse primer (5′-ATTTGAGTATTGAGGGCG-
GGAT3′) with an annealing temperature of 58℃.
PCR amplification was carried out in a PTC-100TM
PCR instrument (MJ Research, Inc., Massachusetts,
USA) with a total reaction volume of 50 μL contain-
ing 150 ng DNA, 5 μL 10× PCR standard reaction
buffer, 10 pmol/L dNTPs, 50 mmol/L MgCl2, 20
pmol/L each forward and reverse primer, 2.5 U Taq
DNA polymerase from Promega (China). Following
an initial denaturation at 95℃ for 3 min, 30 cycles
were performed with 94℃ for 45 s, 58℃ for 45 s,
72℃ for 1 min. The final cycle was followed by an
extension at 72℃ for 10 min. PCR products were
detected on 1.5% agarose gel including 0.5 μg/mL of
ethidium bromide, photographed under UV light, and
sequenced by Bioasia Biological and Technology Co.
Ltd. (Beijing, China).
1. 3 Data analysis
To analyze the sequence variation in the mtDNA
Fig. 1 Repeat structure in sheep mtDNA D-loop region
RⅠ–RⅤrepresent the first through the fifth repeat. T (-) represents with or without an insertion of T between two repeats. The
lines under RⅢ and RⅤ represent deletion of the core tandem repeat sequence in some individuals.
遗传学报 Acta Genetica Sinica Vol.33 No.12 2006
D-loop region, alignment of sequences from 77 sheep
was achieved using BioEdit (Version 5.0.9). DnaSP
(Version 4.0) was used to sort haplotypes and analyze
the number of segregating sites (S), haplotype diver-
sity (Hd)[10], average number of nucleotide differ-
ences (K)[11], nucleotide diversity for each population
(π)[10], genetic distance (Fst)[12] and the genetic dif-
ferentiation (Snn)[13] from nucleotide sequence data
information. The UPGMA program of Phylip (Ver-
sion 3.6) was used to construct the phylogenetic tree
of haplotypes based on polymorphic sites and popula-
tions based on Fst.
2 Results
2. 1 Structure of tandem repeat sequence
The core repeat sequence with a length of 75 bp
is as follows: AATATTAATGTAATATAGACATTAT-
ATGTATAAAGTACATTAAATGATTTACCCCATG-
CGTATAAGCACGTACAT, which was repeated five
times and caused heteroplasmy in different individu-
als. The repeated structure was shown in Fig. 1. There
were five T insertions and 72 gaps between R Ⅰ and
R Ⅱ, R Ⅱ and R Ⅲ, and R Ⅲ and R Ⅳ, which were
all from the same individuals. Three insertions be-
tween RⅣ and RⅤ were also from the same indi-
viduals. R Ⅲ and R Ⅴ were deleted in 4 and 72
individuals, respectively.
2. 2 Polymorphism and haplotypes within the
tandem repeat sequences
A total of 28 variable sites were detected within
309 repeated sequences, among which 7 sites were
singleton variable sites with two variants, 1 site was a
singleton variable site with three variants, and 20
sites were parsimony informative sites with two
variants. A total of 63 haplotypes was sorted using
 LI Xiang-Long et al.: Study on Tandem Repeat Sequence Variation in Sheep mtDNA D-loop Region 1089

DnaSP software (Fig. 2). The proportions of different

haplotypes shown in Table 1 indicate that Hap 1 and
Hap 3 have the highest frequency at 12.94% and
30.42%, respectively, and can be considered as main
or basic haplotypes. The phylogenetic tree among the
haplotypes constructed with the Phylip program is
shown in Fig. 3. All of the haplotypes were sorted
into two main groups except for Hap 14, Hap 60, and
Hap 61. The two basic haplotypes, Hap 1 and Hap 3
are separated in Group Ⅰ with 34 haplotypes and
Group Ⅱ with 26 haplotypes, respectively. Fig. 2
shows that Hap 1 and 3 have significant nucleotide
differences, and the haplotypes with lower propor-
tions in Group Ⅰ were mutated from Hap 1, and
those in Group Ⅱ were mutated from Hap 3.

2. 3 Genetic differentiation of populations

The genetic differentiation (Snn) and the genetic

distances (Fst) among populations are given in Table
2. The UPGMA dendrogram of populations based on
Fst is shown in Fig. 4. Significant Snn (0.2459, P <
0.001) as well as the Fst (0.0767) by permutation test
with 1 000 replicates based on the sequence data in-
formation is obtained. The smaller genetic distance in
Table 2 between Tan sheep and Small Tail Han sheep
(−0.0013), Kazakstan sheep and Altay sheep
(−0.0032), Mongolian sheep and Ujumuqin sheep
(−0.0003) indicates their close relationship (Fig.4).
Meanwhile, the genetic differentiation between Tan
sheep and Small Tail Han sheep (0.5515), Kazakstan
sheep and Altay sheep (0.4817), Mongolian sheep
and Ujumuqin sheep (0.5556) is relatively small and
not significant. Two imported sheep breeds, Dorset
sheep, and Merino sheep, have small genetic distance
(0.0037, Table 2) and a close relationship (Fig. 4),
whereas they differentiate significantly away from the
Chinese indigenous sheep breeds studied here (Table
2). Fig. 2 Haplotypes generated from 309 repeat sequences
2. 4 Genetic diversity of populations of 77 individuals

The number of sequences (N), number of segre- diversity (Hd), average number of differences (K),
gating sites (S), number of haplotypes (h), haplotype and the nucleotide diversity (π ) calculated using
 1090 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006

Table 1 Haplotypes and their distribution in different repeat sequences

Haplotypes No. Percent RⅠ RⅡ RⅢ RⅣ RⅤ Haplotypes No. Percent RⅠ RⅡ RⅢ RⅣ RⅤ
Hap 1 40 12.94 18 12 8 2 Hap 33 1 0.32 1
Hap 2 2 0.65 2 Hap 34 2 0.65 2
Hap 3 94 30.42 30 32 28 4 Hap 35 24 7.77 1 23
Hap 4 2 0.65 2 Hap 36 1 0.32 1
Hap 5 1 0.32 1 Hap 37 1 0.32 1
Hap 6 1 0.32 1 Hap 38 12 3.88 2 10
Hap 7 2 0.65 2 Hap 39 1 0.32 1
Hap 8 5 1.62 3 1 1 Hap 40 2 0.65 1 1
Hap 9 1 0.32 1 Hap 41 13 4.21 13
Hap 10 4 1.29 2 1 1 Hap 42 1 0.32 1
Hap 11 1 0.32 1 Hap 43 1 0.32 1
Hap 12 3 0.97 1 1 1 Hap 44 1 0.32 1
Hap 13 2 0.65 1 1 Hap 45 1 0.32 1
Hap 14 1 0.32 1 Hap 46 2 0.65 2
Hap 15 2 0.65 2 Hap 47 1 0.32 1
Hap 16 4 1.29 2 2 Hap 48 1 0.32 1
Hap 17 5 1.62 1 2 2 Hap 49 1 0.32 1
Hap 18 10 3.24 1 4 5 Hap 50 2 0.65 2
Hap 19 1 0.32 1 Hap 51 1 0.32 1
Hap 20 3 0.97 1 1 1 Hap 52 1 0.32 1
Hap 21 13 4.21 3 5 3 2 Hap 53 2 0.65 2
Hap 22 5 1.62 1 4 Hap 54 1 0.32 1
Hap 23 3 0.97 2 1 Hap 55 1 0.32 1
Hap 24 2 0.65 1 1 Hap 56 2 0.65 2
Hap 25 1 0.32 1 Hap 57 1 0.32 1
Hap 26 1 0.32 1 Hap 58 1 0.32 1
Hap 27 3 0.97 2 1 Hap 59 1 0.32 1
Hap 28 2 0.65 1 1 Hap 60 1 0.32 1
Hap 29 1 0.32 1 Hap 61 1 0.32 1
Hap 30 2 0.65 1 1 Hap 62 1 0.32 1
Hap 31 8 2.59 4 4 Hap 63 1 0.32 1
Hap 32 1 0.32 1 Deletion 76 4 72

DnaSP software are shown in Table 3. For the to-
tal of 309 repeat sequences, 28 segregating sites were
detected. The number of segregating sites and the
number of haplotypes are from 9 to 15 and 7 to 16,
respectively. Small Tail Han sheep and Merino sheep
had the highest and lowest corresponding values (15
and 16; 9 and 7). Kazakstan sheep had the highest
haplotype diversity, average number of differences
and nucleotide diversity (0.9249, 3.0672 and 0.0409),
and Ujumuqin sheep had the lowest corresponding
values (0.6156, 1.7758 and 0.0237). Tibetan sheep
and Mongolian sheep had relatively lower haplotype
diversity and average number of differences and nu-
cleotide diversity than other breeds, whereas Tan
sheep, Hu sheep, and Altay sheep had relative higher
corresponding values.
3 Discussion
3. 1 Origin of Chinese indigenous sheep breeds
Although the origin of Chinese indigenous sheep
breeds has been studied at many levels, it is still un-
 LI Xiang-Long et al.: Study on Tandem Repeat Sequence Variation in Sheep mtDNA D-loop Region 1091

Fig. 3 The UPGMA dendrogram of haplotypes based on polymorphic sites

certain. Ovis arkal, Ovis ammon, or Ovis argal in- Chinese indigenous sheep breeds[14]. Based on the
cluding its subspecies, O. a. darvini and O. a. hadg- differences in head type, horn type, and body con-
soni were considered having a close relationship with struction between Mongolian sheep found on the
 1092 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006

Fig. 4 The UPGMA dendrogram of populations based on Fst

Table 2 Genetic distance (Fst) and genetic differentiation (Snn) among populations
Pop 1 Pop 2 Pop 3 Pop 4 Pop 5 Pop 6 Pop 7 Pop 8 Pop 9 Pop 10
Pop 1 0.6515* 0.5515 0.7056*** 0.6916** 0.8227*** 0.7292*** 0.7251*** 0.7866*** 0.7083***
Pop 2 0.0067 0.6159* 0.6146*** 0.7742*** 0.8241*** 0.7258** 0.6414** 0.6870*** 0.6474**
*** * ** * ***
Pop 3 −0.0013 0.0024 0.7080 0.6155 0.6905 0.6554 0.6911 0.7510*** 0.6345***
Pop 4 0.0661 0.0325 0.0533 0.8459*** 0.8844*** 0.6953*** 0.6601*** 0.6042*** 0.5556
Pop 5 0.0225 0.0395 0.0135 0.1251 0.6899** 0.8438*** 0.8017*** 0.9315*** 0.8431***
Pop 6 0.0210 0.0289 0.0101 0.0990 0.0037 0.9398*** 0.8399*** 0.9896*** 0.9329***
Pop 7 0.0226 0.0109 0.0171 0.0333 0.0635 0.0500 0.4817 0.7048*** 0.6579**
Pop 8 0.0241 0.0079 0.0235 0.0472 0.0644 0.0465 −0.0032 0.7613*** 0.6845***
Pop 9 0.0961 0.0562 0.0817 0.0110 0.1607 0.1319 0.0550 0.0764 0.5678*
Pop 10 0.0686 0.0357 0.0569 −0.0003 0.1376 0.1149 0.0353 0.0505 0.0002
Note: Pop 1-10 are Hu sheep, Tan sheep, Small Tail Han sheep, Tibetan sheep, Dorset sheep, Merino sheep, Kazakstan sheep, Altay
sheep, Ujumuqin sheep, and Mongolian sheep, respectively. Data in the lower triangle and upper triangle are Fst and Snn, respec-
tively. Asterisks *, **, and *** represent P < 0.05, P < 0.01 and P < 0.001, respectively.

Table 3 Genetic diversity among different populations

Breeds N S h Hd K π
Tan sheep 38 11 14 0.9004 2.6572 0.0354
Hu sheep 41 9 15 0.9134 2.6463 0.0353
Small Tail Han sheep 28 15 16 0.8968 2.9603 0.0395
Ujumuqin sheep 45 12 12 0.6156 1.7758 0.0237
Dorset sheep 20 11 10 0.8000 2.8000 0.0281
Merino sheep 13 9 7 0.8462 2.5897 0.0345
Kazakstan sheep 23 12 12 0.9249 3.0672 0.0409
Altay sheep 27 12 13 0.9231 2.7293 0.0364
Tibetan sheep 42 10 8 0.6990 2.0441 0.0273
Mongolian sheep 32 10 9 0.7016 2.1956 0.0293
Total 309 28 63 0.8783 2.7365 0.0365
Note: N, S, h, Hd, K and π represent the number of sequences, number of segregating sites, number of haplotypes, haplotype diver-
sity, average number of differences, and nucleotide diversity, respectively.
 LI Xiang-Long et al.: Study on Tandem Repeat Sequence Variation in Sheep mtDNA D-loop Region
Mongolian plateau and Tibetan sheep on the Tibetan
plateau, Li[15] pointed out that Mongolian sheep and
Tibetan sheep might originate from O. a. daruini and
O. a. hadgsoni of Ovis ammon. In certain articles,
Ovis ammon arkal was also considered as one of the
subspecies of Ovis ammon [16].
It is obvious from Table 1 and Fig. 2 that Hap 1
and Hap 3 are two main and basic haplotypes, from
which other haplotypes mutated. Hap 1 and Hap 3
also were sorted into different groups (Fig. 3). So it
could be inferred that Chinese indigenous sheep
breeds originated from two maternal ancestors be-
cause of the maternal inheritance characteristics of
mtDNA. This result is consistent with that of archae-
ology[16,17], RFLP of mtDNA [7], RAPD[18], and mi-
crosatellite DNA[19]. Two imported sheep breeds,
Dorset sheep and Merino sheep, had the same haplo-
type as Chinese indigenous sheep breeds, indicating
that they had common maternal ancestors. This result
is in accordance with that of Hiendleder et al.[2],
which concluded that domestic sheep were derived
from two different ancestral maternal sources based
on mtDNA analysis. Hiendleder et al. [20] sequenced
and analyzed the complete mtDNA control region
from wild sheep of the mouflon (O. musimon, O.
orientalis), urial (O. vignei), argali (O. ammon) and
bighorn (O. canadensis), and domestic sheep from
Asia, Europe, and New Zealand to investigate wild
sheep taxonomy and the origin of domestic sheep
(Ovis aries), which indicated that domestic sheep
originated from two different subspecies. But the re-
sults of this study are not in agreement with the result
of Guo et al.[9], who found a novel maternal lineage
in Chinese native sheep. Guo et al. [9] pointed that
Chinese sheep breeds are mainly composed of haplo-
types A and B, with a small fraction of haplotype C,
which has not been subject to a recent population
expansion according to Fu’s test and mismatch dis-
tribution. These different results might be caused by
the difference of the sequenced region.
It was also noted that Hap 1 and Hap 3 mainly
exist in the first (R Ⅰ), second (R Ⅱ) and third (R Ⅲ)
repeat sequences. Although Hap 35, Hap 38, and
1093
Hap41 have a relatively high frequency, 7.77%,
3.88%, and 4.21% respectively, they mainly exist in
the fourth repeat sequences (R Ⅳ) (Table 1). It could
be inferred that more variations would appear with
the increase of repeats.
3. 2 Differentiation of Chinese indigenous sheep
breeds
Altay sheep and Kazakstan sheep have a close
relationship (Fig. 4) and a smaller albeit insignificant
genetic differentiation value (0.4817, Table 2). The
genetic differentiation between these two breeds
based on microsatellite DNA was also not signify-
cant[19]. These results are consistent with the opinion
that Altay sheep is one of the advanced local types of
Kazakstan sheep[14]. Similarly, the close relationship
between Mongolian sheep and Ujumuqin sheep (Fig.
4) and their insignificant genetic differentiation
(0.5556, Table 2) are also consistent with Ujumuqin
sheep, which is considered one of the local types of
Mongolian sheep[14].
The close relationship between Mongolian sheep,
Ujumuqin sheep, and Tibetan sheep is not consistent
with the results of cytogenetic analysis [21], RAPD[18],
and microsatellite DNA[19]. Pang et al. [21]
showed
that the relative chromosome length and arm ratio of
Tibetan sheep were different from Mongolian sheep,
Small Tail Han sheep, and Hu sheep. Gong et al. and
Li et al. [18, 19] showed that Tibetan sheep had a distant
relationship with Mongolian sheep; therefore, more
individuals from these sheep breeds should be studied
to clarify this issue.
Ujumuqin sheep, Small Tail Han sheep, and Hu
sheep were generally considered to have originated
from Mongolian sheep[21], a finding that was also
confirmed by RFLP of mtDNA[7]. In this article, only
Ujumuqin sheep had a close relationship with Mon-
golian sheep (Fig. 4) and did not differentiate signifi-
cantly (0.5556, Table 2), whereas Small Tail Han
sheep had a close relationship with Tan sheep (Fig. 4),
a finding consistent with that of RAPD [18]. Mean-
while, Hu sheep and Small Tail Han sheep had a dis-
tant relationship with Mongolian sheep (Fig. 4) and
 1094
differentiated significantly (0.474, P < 0.01 and
0.6345, P < 0.001 respectively, Table 2). This result
indicates that Hu sheep and Small Tail Han sheep
have differentiated significantly from Mongolian
sheep through perennial natural and artificial selec-
tions.
The phylogenetic relationship among different
breeds was constructed based on the Fst representing
the genetic differentiation among populations. The
discordant relationship between different breeds
needs to be studied further using more genetic mark-
ers and different methods.
3. 3 Genetic diversity among Chinese indigenous
sheep breeds
Results of genetic diversity among Chinese in-
digenous sheep breeds obtained from different stud-
ies are largely consistent. In this article, Ujumuqin
sheep, Tibetan sheep, and Mongolian sheep have
relatively lower haplotype diversity and average
number of differences and nucleotide diversity com-
pared with other breeds, whereas Kazakstan sheep,
Tan sheep, Hu sheep, and Altay sheep have relatively
higher corresponding values. This result is basically
consistent with that of microsatellite DNA[19], which
showed that Mongolian sheep and Ujumuqin sheep
had lower expected heterozygosity and higher Fis,
which represented decreased genetic diversity, in
contrast to Altay sheep and Kazakstan sheep which
had higher expected heterozygosity and lower Fis. It
could be concluded that Tibetan sheep, Mongolian
sheep, and Ujumuqin sheep have lower genetic diver-
sity, whereas Altay sheep and Kazakstan sheep have
higher genetic diversity.
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绵羊线粒体 DNA D-loop 区串联重复序列变异研究

李祥龙1, 巩元芳2, 刘铮铸2, 郑桂茹1, 周荣艳1, 靳晓敏2, 李兰会1, 王海亮1
1. 河北农业大学动物科技学院，保定 071001；
2. 河北科技师范学院动物科学系，昌黎 066600

摘 要: 对来自 69 个我国地方绵羊品种和 8 个国外引入品种共计 77 个个体线粒体 DNA 控制区长度为 75 bp 的串联重复序

列进行了测序分析。在 309 个重复序列中检测到 28 个变异位点，其中 7 个为具有 2 个变异体的单现突变，1 个为具有 3 个
变异体的单现突变，20 个为具有 2 个变异体的简约位点。由 28 个变异位点中归纳出 63 个单倍型，其中单倍型Ⅰ和单倍型
Ⅲ具有较高的比例，分别为 12.94% 和 30.42%。研究结果揭示我国地方绵羊可能起源于两个母系祖先。哈萨克羊和阿勒泰
羊间以及蒙古羊和乌珠穆沁羊间分别具有较近的亲缘关系且没有明显的遗传分化。藏绵羊、蒙古羊和乌珠穆沁羊相对哈萨
克羊和阿勒泰羊而言具有较低的遗传多样性。
关键词: 绵羊；线粒体 DNA D-loop；串联重复序列；变异

作者简介: 李祥龙（1963-），男，河北丰南人，博士，教授，博士生导师，研究方向: 动物分子育种。

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