全 文 ：第 49 卷 第 11 期
2 0 1 3 年 11 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 49，No. 11
Nov．，2 0 1 3
doi:10．11707 / j．1001-7488．20131114
Received on Aug． 25，2012;Revised on Aug． 27，2013．
Foundation project: National Nature Science Foundation of China (31040018) ; Nature Science Foundation of Shanxi Province(2012011034-3) ;
Shanxi Teaching Reform Project ( J2011088) ．
* We thank Prof． Zhang Longsheng，Prof． Wu Jianyong and the staff at Taiyuan Zoo for providing the blood and feather samples for this study．
褐马鸡圈养种群 ISSR标记的遗传多样性*
武玉珍1 冯睿芝2
(1．晋中学院生物科学与技术学院 晋中 030600; 2． 复旦大学生物医学研究院 上海 200032)
摘 要: 利用 ISSR 分子标记技术对褐马鸡庞泉沟国家自然保护区与太原市动物园 2 个种群的遗传多样性进行
研究。从 20 个 ISSR 引物中筛选出 10 个引物对褐马鸡 2 个种群的 35 个样品进行扩增，得到 65 个清晰的扩增位
点，多态带百分率(PPB)为 76. 9%。2 个种群的多态带百分率和群体内遗传多样性指数分别为 54. 10%，54. 10%，
0. 302 5，0. 205 6，前者遗传多样性略高于后者。2 个种群间的遗传分化系数(G st)、基因流估计值(Nm )、遗传相似性
系数以及遗传距离均表明种群间的遗传分化较小。褐马鸡 UPGMA 系统树无明显的歧化，尚未表现出遗传趋异。
褐马鸡 2 个种群间的遗传多样性指数比较接近，遗传多样性水平较低，核基因组与过去线粒体基因组遗传多样性
的研究结果基本一致。
关键词: 褐马鸡; 濒危物种; 圈养; ISSR; 遗传多样性
中图分类号: S718. 63 文献标识码: A 文章编号: 1001 － 7488(2013)11 － 0103 － 06
ISSR Analysis of Genetic Diversity of Two Brown-Eared
Pheasant (Crossoptilon mantchuricum) Populations
Wu Yuzhen1 Feng Ruizhi2
(1. School of Biology Science and Technology，Jinzhong University Jingzhong 030600; 2. Institute of Biomedical Sciences，Fudan University Shanghai 200032)
Abstract: Brown-eared Pheasant ( Crossoptilon mantchuricum) is a critically endangered endemic species，which is
listed in the national first-class protected birds in China． In this study，ISSR markers were used to investigate the genetic
diversity of two populations of Brown-eared Pheasant in the Pangquangou Nature Reserve and Taiyuan Zoo． Ten primers
out of 20 ISSR primers were selected for amplifying 35 samples of the two populations，and 65 clear amplification sites
(PPB = 76. 9% ) were obtained． The percentage of polymorphic bands of the two populations was around 54. 10% ．
Shannon’s Information Index of the two populations was 0. 302 5 and 0. 205 6，respectively． The generic heterogeneity
(G st)，genetic flow (Nm)，genetic similarity index indicated that there was no significant genetic differentiation between
the two populations． Statistical analysis and cluster tree diagram consistently illustrated that the genetic diversity between
the two populations was at a low level． Individuals in each population had close genetic relationship． The result in this
study were consistent to another study we did with the mitochondrial genome genetic diversity．
Key words: Brown-eared Pheasant; endangered species; populations; ISSR; genetic diversity
ISSRs ( inter-simple sequence repeats) are DNA
fragments amplified from PCR with microsatellite
primers，and can be used as molecular markers for the
different size of anchored primers ( Zietkiewicz et al．，
1994; Gilbert et al．，1999; Fang et al．，1997) ． For
the multi-locus fingerprinting profiles obtained， the
applications of ISSR analysis widely spread to genetic
identity(Aytekin et al．，2011;Noroozi et al．，2011)，
parentage，clone and strain identification，taxonomic
studies on closely related species ( Parsaeian et al．，
2011; Zhao et al．， 2008 )， gene mapping studies
(Arcade et al．，2000; Casasoli et al．，2001; Sanker，
2001; Jin，2007; Hizer et al．，2002; Ostberg et al．，
2002) and genetic relationship judgments (Huang et
林 业 科 学 49 卷
al．，2000; Pharmawati et al．， 2005; Sica et al．，
2005) ． In the studies on animals，ISSRs have been
successfully used in genetic diversity and genetic
structure researches ( Aytekin et al．，2011; Abbot，
2001; Willis et al．，2004; Kostia et al．，2000 ) ． In
China，ISSRs were applied for germplasm analysis of
Eriocheir sinensis and Antheraea pernyi ( Zheng et al．，
2007; Li et al．，2007 ) ． Comparatively fewer studies
using ISSRs for vertebrate have been reported，
especially for critically endangered species． At
present，Bai et al． (2001) and Bai (2004) have used
ISSRs for the fingerprint analysis of captive populations
of Manchurian Tiger and populations of chicken． In
other countries，Aytekin et al． (2011) used ISSRs to
assess genetic diversity in buffalo populations，Kostia
et al． (2000) used ISSR to study the phylogenesis of 6
species of mammal，and Willis et al． ( 2004 ) used
ISSRs and mitochondria DNA markers to study the
natural hybridization of Phocoenoides dalli and
Phocoena phocoen． In summary， the application of
ISSRs in the study of higher animals，especially in the
genetic diversity of endangered species is limited．
In this article，ISSRs together with the identification
results of mitochondria DNA(we reported that in a separate
paper)were used to study the genetic structure and genetic
diversity of two populations of the Brown-eared Pheasant in
Pangquangou Nature Reserve and Taiyuan Zoo． The degree
concerning the genetic differentiation and gene flow
between and within the two populations were also
investigated． The molecular endangering mechanism was
discussed and the genetic diversity was analyzed from
the nuclear DNA level，hopefully that we can provide
nuclear DNA proofs for the protection of the Brown-
eared Pheasant and better understands the survival
potential and the degree of endangering of this bird so as
to assist the formulation and implementation of the
Brown-eared Pheasant protection strategy．
1 Materials and methods
1. 1 Samples
35 samples of the Brown-eared Pheasant from the
two populations were collected， including 3 muscle
samples and 15 blood samples from Pangquangou
Nature Reserve and 17 blood samples from Taiyuan
Zoo，respectively．
1. 2 Genome DNA extraction
Genome DNA was extracted following the method
described by Wu et al．(2010) ． Purity and concentration
of DNA were tested by bio-photometer ( Eppendorf) ．
The DNA samples were diluted to 20 ng·mL － 1 and
stored at － 20 ℃ ．
1. 3 Primer synthesis
The synthesis of primers was conducted by Aoke
Co，Ltd，Beijing following the ISSR primer sequences
UBC set 9． According to the references of the study
results of Abbot et al． (2001) and Bai et al．(2001)，
20 primers were selected． 4 samples were randomly
used as template and 10 primers with stable and clear
PCR amplified bands were chosen to use in all the PCR
with all of the DNA template samples．
1. 4 ISSR-PCR and electrophoresis
PCR was performed in a 25 mL react system
containing 2. 5 mL of 10 × PCR Buffer ( TaKaRa，
China)，2 mL of 25 mmol·L － 1 Mg2 + solution，4 mL of
1. 25 mmol·L － 1 dNTP solution，5. 0 mL of 5 mmol·
L － 1 each primer solution，0. 5 mL of 2 U·mL － 1 Taq
DNA Polymerase (TaKaRa，China) and 1 mL of 10 －
150 ng·mL － 1 DNA template solution． A PTC-150
thermal cycler was used and the thermal cycling
profiles were as follows: an initial hot-start for 5 min at
94 ℃ ; 40 cycles amplification cycles of denaturation
for 45 s at 94 ℃，annealing for 45 s at 38 － 54 ℃ and
extension for 1 min at 72 ℃，and a final incubation for
7 min at 72 ℃ was performed to ensure complete
extension of the PCR． The annealing temperatures of
each primer are showed in Tab． 2． The PCR products
were separated by the 1. 5% agarose gel． DNA
marker: DL2000 (TaKaRa，China) ．
1. 5 Statistical analysis
The primary ( 0，1 ) ISSR analysis matrix was
formulated from the results of the experiments，1 for
products generated and 0 for no products generated．
Percentage of polymorphic band ( PPB)，Shannon’s
Information Index ( I)，Nei’s gene diversity ( H e )，
the total genetic diversity ( H t )，the genetic diversity
within a population ( H s )， the coefficient of gene
differentiation(G st) and the estimate of gene flow(Nm )
were calculated by POPGEN 1. 31(Yeh et al．，1997) ．
Nei’s unbiased genetic distance and genetic similarity
were calculated by PhyTool 6. 0． According to Nei’s
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第 11 期 武玉珍等: 褐马鸡圈养种群 ISSR 标记的遗传多样性
genetic distance， cluster analysis using Neighbor-
Joining Method was performed using MEGA3. 1 and
clustering tree diagram was constructed． Based on
these results，genetic diversity analysis between the two
populations and genetic similarity analysis between
each individual of the two populations were made．
2 Results and analysis
2. 1 PCR-ISSR analysis
PCR for the 35 samples were performed using 10
selected primers ( Tab． 1) ． The results of agarose gel
electrophoresis showed that TA，AG，CT，CA have high
Tab． 1 Distribution of polymorphism loci in the primers
Primer
Primer
sequence
Number of
bands
Number of
polymorphic bands
806 (TA) 8 G 2 － 8 7
807 (TA) 8 G 3 － 8 6
808 (AG) 8 C 2 － 6 5
809 (AG) 8 G 3 － 9 7
811 (GA) 8 C 2 － 8 7
812 (GA) 8 A 2 － 5 3
815 (CT) 8 G 1 － 5 4
816 (CA) 8 T 2 － 6 4
817 (CA) 8 A 1 － 5 4
818 (CA) 8 G 2 － 5 3
Total 65 50
distribution frequency that they have been amplified
from primers with these dinucleotide．
2. 2 Percentage of polymorphic band
The results of amplification using 10 primers were
showed in Tab． 2． It was shown that 65 stable and
clear bands were amplified and the sizes of these
fragments were in the range of 300 － 2 000 bp． The
numbers of amplified loci of each primer was 5 － 9，
averaged 6. 5． Of the total 65 loci 50 loci were
polymorphic; the percentage of polymorphic band was
76. 9% ． Different primers have different numbers of
amplified loci; the percentage of polymorphic band was
60. 00% － 77. 78% ．
2. 3 Genetic diversity of the two populations
Statistical analysis of 35 individuals of the two
populations was performed using POPGRNE1. 31． The
results were showed in Tab． 2． Effective numbers of
alleles，H e and I of the two populations of Pangquangou
Nature Reserve and Taiyuan Zoo are 1. 362 8，
0. 205 6，0. 302 5 and 1. 294 9，0. 175 2，0. 265 9，
respectively． Observed numbers of alleles，number of
polymorphic bands and percentage of polymorphic
bands for the two populations are the same: 1. 541 0，
33，and 54. 10．
Tab． 2 Statistical analysis of genetic diversity in two populations①
Population
Number of
samples
N a N e H e I
Number of
polymorphic bands
PPB
Pangquangou 18 1. 541 0 1. 362 8 0. 205 6 0. 302 5 33 54. 10
Taiyuan Zoo 17 1. 541 0 1. 294 9 0. 175 2 0. 265 9 33 54. 10
Average 17. 5 1. 541 0 1. 328 8 0. 190 4 0. 284 2 33 54. 10
Species 35 1. 623 0 1. 403 6 0. 228 1 0. 336 7 38 62. 30
①N a = Observed number of alleles; N e = Effective number of alleles; H e = Nei’s (1973) gene diversity; I = Shannon’s information index; PPB =
Percentage of polymorphic loci．
At the population level，H e， I and PPB are
0. 190 4，0. 284 2 and 54. 10% ． At the species level，
H e，I and PPB are 0. 228 1，0. 336 7 and 62. 30% ．
Therefore，the genetic diversity at the species level is
higher than that at the population level． The genetic
diversity at population level of Taiyuan Zoo is a little
different from that of Pangquangou Nature Reserve，but
they did not reach the statistical significant level．
Therefore，the genetic diversity of the two populations
is close to each other．
2. 4 Relationship of genetic diversity of
each population
We used POPGRNE1. 31 to calculate genetic
diversity index between and within the two populations．
The H s，H t，G st and Nm are 0. 227 7，0. 190 4，
0. 164 0 and 2. 549 3． On the whole，H s are a little
higher than H t，and H t is at a low level． G st is small，
16. 40% variations between the two populations，
83. 60% variations within the populations and gene
flows exist between them． From Tab． 3， the Nei’s
gene identity of the two populations is 0. 907 9 and the
genetic distance is 0. 096 6． It further illustrates that
the genetic diversity of the two populations are nearly
the same and the genetic variation between them are
not evident．
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林 业 科 学 49 卷
Tab． 3 Nei’s genetic identity and genetic distance
between two populations①
Population 1 2
1 0. 907 9
2 0. 096 6
① Nei s genetic identity ( above diagonal) and genetic distance
( below diagonal) ．
2. 5 Genetic distance and cluster analysis
among individuals
Nei’s genetic distance and genetic similarities of
the two populations between each individual were
calculated using PhylTools software． The average
genetic similarity of the two populations is 0. 506 1 and
0. 759 1，and the average genetic distance is 0. 493 9
and 0. 240 9 respectively． The total of Nei’s genetic
distance and similarity coefficient of average of Brown-
eared Pheasant 35 individuals of the two populations
are 0. 372 3 and 0. 631 0 respectively． Using UPGMA
(unweighted pair-group method with arithmetic means)
Method to perform Nei’s genetic distance cluster
analysis achieved a result showing in Fig． 1，which
demonstrates that the individuals of the two populations
did not form 2 branches，but made cross correlations in
each other’s population．
Fig． 1 Dendrogram of Brown-eared Pheasant based on Nei’s distance of ISSR marker and
clustered using UPGMA dendrogram using average linkage ( between groups)
dwy:Taiyuan Zoo; pqg:Pangquangou
3 Discussions and conclusion
3. 1 ISSR genetic diversity analysis of the Brown-
eared Pheasant
The statistical result of the two population’s shows
that the diversity at species level is higher than that at
population level，and the genetic diversity index of the
two populations are close to each other，which means
the genetic diversity of them is at a low level． At
present，there are fewer reports in China using ISSR on
birds and there is no comparison data for the study．
However， the results are consistent with that using
mitochondria control region as molecular marks in our
previous publication (Wu et al．，2010) ．
There is truly different between the genetic
diversity of the two populations although not
significant． The genetic diversity of the population of
Pangquangou Nature Reserve is a little higher than that
of Taiyuan Zoo ( I of them are 0. 302 5 and 0. 205 6，
and H e of them are 0. 265 9 and 0. 175 2) ．
As ISSR is dominant marker，we cannot tell that
related loci are homozygous or heterozygous． Therefore
the ignorance of heterozygous gene will contribute to
H e，which leads to the underestimate to I for the total
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第 11 期 武玉珍等: 褐马鸡圈养种群 ISSR 标记的遗传多样性
genetic diversity and the genetic diversity within the
population． The results in this research can be used to
compare with other co-dominant molecular markers
such as SSR and AFLP in further study，and enlarging
sample range is another important aspect of better
understanding of genetic diversity of the Brown-eared
Pheasant．
3. 2 Analysis of genetic variations between the two
populations
The genetic diversity of the Brown-eared Pheasant
populations is at a low level． The two populations have
nearly the same genetic diversity within the population，
which is higher than genetic diversity between the two
populations． Genetic differentiation between the two
populations is lower and there are gene flows． Professor
Wright ( Wright， 1931 ) thought that if gene flow
between populations is ＞ 1，then homogenization will
be made． Conversely，if gene flow is ＜ 1，then it will
be the main reason for genetic differentiation． In our
research，gene flow between the two populations is
＞ 1. 16. 40% variations are between the two
populations and 83. 60% variations are within the
populations． Genetic similarity of the two populations is
0. 907 9 and genetic distance is 0. 096 6，
demonstrating that the genetic diversity levels of the
two populations are consistent， and genetic
differentiation between them is not significant mainly
due to the introduction of the species of Taiyuan Zoo
from Pangquangou Nature Reserve．
3. 3 Comparison between ISSR markers and
mitochondria gene markers
The polymorphism of mitochondria DNA and
nuclear DNA are sometimes inconsistent ( Moritz，
1991; Curole et al．，1999) ． Different genetic diversity
indexes were obtained by using ISSR markers and
mitochondria control region． However，both of them
showed that the genetic diversity of the Brown-eared
Pheasant of the two populations did not reach the
statistical significant level and they are consistent．
For mitochondria control region， the average
Nei’s genetic distance of the individuals of the
population of Pangquangou Nature Reserve and Taiyuan
Zoo are 0. 002 and 0. 003，respectively． The average
Nei’ s genetic distance of 20 individuals from
Pangquangou Nature Reserve and Taiyuan Zoo is 0. 002
(max: 0. 009; min: 0. 000) ． For ISSRs，the average
Nei ’ s genetic similarity of individuals from
Pangquangou Nature Reserve and Taiyuan Zoo are
0. 506 1 and 0. 759 1， respectively． The average
genetic distance of them is 0. 493 9 and 0. 240 9，
respectively． The average Nei’s genetic distance and
genetic similarity of the whole 35 individuals from the
two populations are 0. 372 3 and 0. 631 0，respectively
(max: 1. 000; min: 0. 000) ． The average of Nei’s
gene diversity between the two populations is 0. 907 9，
and the average genetic distance is 0. 372 3． Although
above indexes are different，the genetic differentiation
between the two populations of the two places is at a
low level and the genetic variation between individuals
of the two populations is not significant，suggesting that
the individuals have close genetic relationship．
3. 4 Conclusions
These data reflect the low level of genetic diversity
of the Brown-eared Pheasant populations． The results
of nuclear genome and mitochondria genome are
consistent and both prove that the genetic
differentiation between the two populations is not
significant． Individuals between the two populations
have close genetic distance and high degree of
inbreeding．
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(责任编辑 朱乾坤)
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