全 文 ：基于 ISSR 分子标记的新疆欧
洲李亲缘关系分析
孙琪，廖康 *，耿文娟，刘娟，曼苏尔·那斯尔，刘
欢，贾杨，曹倩 （新疆农业大学特色果树研究
中心，新疆乌鲁木齐 830052）
摘 要 [目的] 探究不同新疆欧洲李种质类
型间的亲缘关系。 [方法] 运用 ISSR 分子标记
对 5 个类型共 30 个单株的新疆欧洲李种质资
源进行遗传多样性及亲缘关系分析 。 [结果 ]
筛选获得的 16 条引物共扩增出 317 条带，其
中有多态性条带 246 条 ， 占扩增总数的
77.60%。 供试单株的 Nei’s 遗传多样性指数
（H）为 0.266 6，平均 Shannon 信息多样性指数
（I） 为 0.399 1， 遗传相似系数多在 0.555 2~
0.996 8 之间， 表明新疆欧洲李资源具有一定
的遗传多样性。 聚类分析显示，在遗传相似系
数 0.719 处 30 株供试欧洲李被划分为 3 个大
组，国外引进欧洲李品种一组、塔城酸梅和塔
城槟子一组、 伽师酸梅和野生欧洲李一组，而
在相似系数 0.949 处，伽师酸梅和野生欧洲李
又分为两个小组。 [结论] 伽师酸梅与野生欧
洲李的亲缘关系很近，国外引进欧洲李品种与
塔城酸梅及塔城槟子的亲缘关系较近。
关键词 新疆；欧洲李；ISSR；亲缘关系
基金项目 野生欧洲李亲缘关系研究；国家自
然科学基金项目（31160387）；新疆维吾尔自治
区果树重点学科基金。
作者简介 孙琪（1989-），女，新疆乌鲁木齐人，
在读硕士研究生，研究方向：果树种质资源 ，
E-mail：1056441863@qq.com。 *通讯作者，教授，
博士生导师，E-mail：13899825018@163.com。
收稿日期 2014-12-22
修回日期 2015-02-03
Analysis of Genetic Relationships in Prunus
domestica L. in Xinjiang Using ISSR Markers
Qi SUN, Kang LIAO*, Wenjuan GENG, Juan LIU, Mansur NASIR, Huan LIU, Yang JIA, Qian CAO
Research Centre of Characteristic Fruit Tree, Xinjiang Agricultural University, Urumqi 830052, China
Supported by Study on Genetic Relationships of Wild European Plum; National Natural
Science Foundation of China (31160387); Financial Aid from Xinjiang Uygur Autono-
mous Region Fruit Trees Key Subject.
*Corresponding author. E-mail: 13899825018@163.com
Received: December 22, 2014 Accepted: February 3, 2015A
Agricultural Science & Technology, 2015, 16(3): 447-453, 503
Copyright訫 2015, Information Institute of HAAS. All rights reserved Molecular Biology and Tissue Culture
A t the taxonomy, Prunus do-mestica L. belongs to Prunus,Rosaceae[1]. There are several
types of P. domestica germplasm re-
sources across Xinjiang. For example,
there are Jiashi Smoked Plum and in-
troduced European plum varieties in
Kashgar, Wild European Plum in Ili,
and Tacheng Smoked Plum and
Tacheng Binzi in Tacheng, etc [2]. Xin-
jiang is the only region where Wild
European Plum has been discov-
ered [3 -4]. The origin of P. domestica is
still controversial. Someone considers
that P. domestica is the offspring
formed by crossing between Prunus
cerasifera L. and Prunus spinosa L.;
while someone considers that Wild
European Plum which is only distribut-
ed in Xinjiang is the true progenitor of
cultivated P. domestica [5-9]. This study
clarified the genetic relationships
among different types of P. domestica
germplasm resources in Xinjiang so as
to help to explore the genetic charac-
teristic of P. domestica germplasm re-
sources in depth and to lay a founda-
tion for future studies on origin and
new variety breeding of P. domestica.
There are rare previous studies
on P. domestica at the molecular level.
Geng et al.[10] analyzed the genetic re-
lationships of P. domestica by SSR
markers. They found the genetic diver-
sity in the internal of Wild European
Plum was limited, and the one who
had closest genetic relationship with
Wild European Plum was local vari-
eties. Liu [5], Feng [11] and Liang et al.[12]
all involved parts of P. domestica ma-
terials when they studied Prunus and
its related species, and they speculat-
ed that there was a closer genetic re-
lationship between P. domestica and
Abstract [Objective] This paper aimed to explore the genetic relationships among
different types of Prunus domestica L. in Xinjiang. [Method] The inter-simple se-
quence repeat (ISSR) markers were applied to analyze the genetic diversity and re-
lationships among 30 individual plants from 5 types of P. domestica germplasm re-
sources in Xinjiang. [Result] A total of 317 bands were amplified by 16 selected IS-
SR primers. Among the amplified bands, there were 246 polymorphic bands, ac-
counting for 77.60% of the total. The Nei’s gene diversity index (H) of individuals
was 0.266 6. The average Shannon’s information index (I ) of individuals was 0.399 1.
And most of the genetic similarity coefficients ranged from 0.555 2 to 0.996 8. It is
indicated that the P. domestica germplasm resources have a certain genetic diversi-
ty in Xinjiang. The cluster analysis showed that, at the genetic similarity coefficient
of 0.719, the 30 individuals were divided into 3 major groups, including a group of
introduced European plum varieties, a group of Tacheng Smoked Plum and
Tacheng Binzi and a group of Jiashi Smoked Plum and Wild European Plum. While
at the genetic similarity coefficient of 0.949, Jiashi Smoked Plum and Wild European
Plum were divided into two small groups. [Conclusion] There are very close genetic
relationships between Jiashi Smoked Plum and Wild European Plum, and among
the introduced European plum varieties, Tacheng Smoked Plum and Tacheng Binzi.
Key words Xinjiang; Prunus domestica L.; ISSR; Genetic relationship
DOI:10.16175/j.cnki.1009-4229.2015.03.009
Agricultural Science & Technology 2015
P. spinosa. Decroocq et al. [13] studied
the genetic relationship between par-
ent and offspring of introduced culti-
vated P. domestica by using chloro-
plast cDNA-SSR markers. Shimada et
al.[14] investigated the genetic relation-
ships among Prunus plants by using
RAPD markers, and hypothesized
that there might exist a possibility of
crossing between P. domestica and
P. salicina.
ISSR is considered an efficient,
reliable and highly polymorphic
molecular marker[15]. But so far, there
has been no research available on the
genetic relationships among different
types of P. domestica germplasm re-
sources in Xinjiang by using ISSR
markers. Therefore, this study select-
ed Jiashi Smoked Plum, introduced
European plum varieties, Wild Euro-
pean Plum, Tacheng Smoked Plum
and Tacheng Binzi as the test materi-
als and studied the genetic character-
istics and relationships of representa-
tive types of P. domestica germplasm
resources in Xinjiang using ISSR
markers.
Materials and Methods
Materials
The 30 tested individual plants of
P. domestica, including 6 individuals of
Jiashi Smoked Plum, 4 varieties of in-
troduced European plum, 8 individuals
of Tacheng Binzi, 6 individuals of
Tacheng Smoked Plum and 6 individ-
uals of Wild European Plum were
growing in 4 regions (Jiashi, Luntai,
Tacheng and Ili) of Xinjiang. The spe-
cific distribution sites and types of sam-
pled individuals were listed in Table 1.
Methods
Sampling After leaves were expand-
ed in spring, 30 to 50 pieces of tender
leaves without petioles were collected
from each of the individual plants, re-
spectively. The sampled leaves were
placed into sealed pockets with silica
until they were dried completely. Sub-
sequently, the dried leaves were fully
grinded in liquid nitrogen, and the
leaves powders were collected into
centrifuge tubes and stored at -80℃.
Extraction of DNA and establish-
ment of ISSR polymerase chain
reaction procedure The DNAs of
P. domestica individual plants were
extracted by using modified CTAB
method[16]. The purities and concentra-
tions of extracted DNAs were exam-
ined with 0.8% agarose gel elec-
trophoresis and ultraviolet spec-
trophotometer. The values of OD230,
OD260 and OD280 of each sample were
recorded. Then the extracted DNA
was diluted into concentrations of 50 to
80 ng/μl. Referring to the orthogonal
and single factor experimental design
methods of Bai et al. [17], Wang[18] and
He et al. [19], the 20 μl ISSR-PCR pro-
cedures were optimized. The finally
optimized amplification system (20 μl)
was as follows: 1.0 U Taq polymerase,
0.75 μmol/L primer, 0.25 mmol/L
dNTPs, 2.0 mmol/L Mg2 + , 50 -80 ng
DNA, 2 μl 10× Buffer and double dis-
tilled water. The optimized amplifica-
tion conditions were as follows: pre-
denaturation at 94 ℃ for 4 min; denat-
uration at 94 ℃ for 4 s, annealing at
50-63 ℃ (depending on primers) for
1min, extension at 72 ℃ for 2 min, 33
cycles; final extension at 72 for 7 min.
The PCR amplifications would pro-
ceed in a gradient thermal cycler
(TProfessional, Biometra). The ampli-
fied products were examined by 2.0%
agarose gel electrophoresis in 1× TAE
buffer at 120 V and 165 mA for 35 min
(DYY-7C, Liuyi (Beijing)). The gels
were stained with 10 mg/ml ethidium
bromide for 15 min and then pho-
tographed under the ultraviolet light gel
imaging system (Alphalmager HP).
The used molecular weight standard
was Trans2K DNA Marker. The Taq
polymerase, dNTPs, Mg2+, Buffer and
Marker were all purchased from the
Beijing Transgen Biotech Co., Ltd.
Selection of primers and their an-
nealing temperatures Referring to
related researches[5, 11, 20-23], the 59 IS-
SR primers for amplifying Prunus
sequences were selected and then
synthesized by the Beijing Genomics
Institute. The individuals of JS-1 and
YE-1, which had big differences in ap-
pearance, were used to select primers.
To optimize the annealing tempera-
ture, a total of 8 gradient annealing
temperatures (recommended anneal-
ing temperature -3, -2, -1, 0, +1, +2,
+3, +4 ℃ ) were designed for each of
the primers. The primers and its opti-
mum annealing temperatures which
had richer and brighter bands, cleaner
background and more stable amplifi-
cation were finally selected.
ISSR markers The ISSR markers of
genomic DNAs of the 30 individual
plants were performed with selected
primers and their optimum annealing
temperatures under conditions of opti-
mum amplification system, optimum
amplification conditions and optimum
cycle index. The amplified bands of
each primer for each individual were
counted, respectively. At certain site of
certain lane, if there is one band, it is
recorded as ‘1’; if there is no band, it
is recorded as ‘0’. At final, a matrix
composed of 0 and 1 was obtained for
each sample.
Data statistics The obtained data
was first processed with Excel 2010
and then analyzed by using SPSS
20.0. The total numbers of amplified
bands and polymorphic bands were
counted, and then the ratios between
them were calculated. POPGEN3.2
was used to calculate the Nei’s gene
diversity index (H), Shannon’s infor-
mation index (I) and other kinds of
similarity coefficients and genetic in-
dexes. NTSYSPC2.1 was used to
draw the dendrogram.
Results and Analysis
Selection of primers and their opti-
mum annealing temperatures
At final, a total of 16 primers,
along with their optimum annealing
temperatures which had richer and
clearer bands, cleaner background
and more stable amplification were
screened out from 59 ISSR primers
(Table 2). There were 11 primers of
which the optimum annealing temper-
atures were higher than the recom-
mended annealing temperatures;
there were 2 primers of which the opti-
mum annealing temperatures were as
same as the recommended annealing
temperatures; there were 3 primers of
which the optimum annealing temper-
atures were lower than the recom-
mended annealing temperatures. The
results showed that there were only 2
primers of which the optimum anneal-
ing temperatures were same with the
recommended annealing tempera-
tures. It is indicated the exploration of
optimum annealing temperatures is
necessary and essential.
YE-1 was used as the template. It
was amplified with UBC836 and
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Agricultural Science & Technology2015
Table 1 Types and distribution sites of sampled individuals
Serial
No. No. Latitude∥N° Longitude∥E° Altitude∥m
Germplasm type and
variety Sampling site
1 JS-2 39.499 881 76.840 031 1 201.50 Local variety (Jiashi
Smoked Plum)
Ardila Village, Yingmaili Township, Jiashi
Country
2 JS-6 39.501 888 76.820 999 1 206.50 Local variety (Jiashi
Smoked Plum)
Beside the villagers committee of Village 8,
Yingmaili Township, Jiashi Country
3 JS-12 39.508 001 76.778 923 1 218.40 Local variety (Jiashi
Smoked Plum)
Demonstration Garden of Agro-Tech Promotion
and Service Center of Jiashi Country
4 JS-13 39.515 585 76.773 653 1 213.10 Local variety (Jiashi
Smoked Plum)
Group 2, Village 20, Township 1, Jiashi Country
5 JS-14 39.514 636 76.773 926 1 203.50 Local variety (Jiashi
Smoked Plum)
Group 2, Village 20, Township 1, Jiashi Country
6 JS-15 39.506 468 76.781 456 1 210.60 Local variety (Jiashi
Smoked Plum)
Group 2, Village 20, Township 1, Jiashi Country
7 JS-1 39.499 954 76.840 107 1 196.40 Introduced European
plum (Goddess)
Ardila Village, Yingmaili Township, Jiashi
Country
8 JS-3 39.499 879 76.840 021 1 199.20 Introduced European
plum (French)
Ardila Village, Yingmaili Township, Jiashi
Country
9 JS-4 39.499 869 76.840 045 1 200.90 Introduced European
plum (Stanley)
Ardila Village, Yingmaili Township, Jiashi
Country
10 L1 41.806 808 84.260 872 971.20 Introduced European
Plum (Richard)
Plantation of Seed Multiplication Farm of Luntai
Country
11 T2 46.924 380 83.113 192 851.50 Local variety (Tacheng
Binzi)
Farm of Water Management Institute of
Tacheng
12 T3 46.924 842 83.132 231 857.10 Local variety (Tacheng
Binzi)
Farm in BangkokNoor Village, Asir Township,
Tacheng
13 T4 46.924 863 83.132 288 855.40 Local variety (Tacheng
Binzi)
Farm in BangkokNoor Village, Asir Township,
Tacheng
14 T5 46.889 977 83.067 224 811.20 Local variety (Tacheng
Binzi)
Farm in Group 2, Qianwei Village, Asir
Township, Tacheng
15 T6 46.938 377 83.104 585 881.10 Local variety (Tacheng
Binzi)
Farm in Group 3, Qianwei Village, Asir
Township, Tacheng
16 T7 46.733 864 82.962 710 544.40 Local variety (Tacheng
Binzi)
Farm house nearby the 5th Middle School of
Tacheng
17 T9 46.733 883 82.962 750 540.40 Local variety (Tacheng
Binzi)
Farm house nearby the 5th Middle School of
Tacheng
18 T12 46.713 269 82.946 872 518.60 Local variety (Tacheng
Binzi)
Plantation of Agricultural Research Institute of
Tacheng
19 T1 46.733 741 82.962 712 533.50 Local variety (Tacheng
Smoked Plum)
Farm house nearby the 5th Middle School of
Tacheng
20 T8 46.733 869 82.962 726 543.00 Local variety (Tacheng
Smoked Plum)
Farm house nearby the 5th Middle School of
Tacheng
21 T10 46.710 946 82.947 698 514.70 Local variety (Tacheng
Smoked Plum)
Plantation of Agricultural Research Institute of
Tacheng
22 T11 46.710 896 82.947 476 518.50 Local variety (Tacheng
Smoked Plum)
Plantation of Agricultural Research Institute of
Tacheng
23 JS-5 39.501 876 76.820 973 1 208.00 Local variety (Tacheng
Smoked Plum)
Beside the villagers committee of Village 8 in
Yingmaili Township, Jiashi Country
24 JS-11 39.507 524 76.779 507 1 223.00 Local variety (Tacheng
Smoked Plum)
Demonstration Garden of Agro-Tech Promotion
and Service Center of Jiashi Country
25 YE-1 43.500 192 83.696 679 1 299.80 Wild resource (Wild
European Plum)
Mountains in Birsay, Xinyuan Country, Ili
26 YE-2 43.500 387 83.696 988 1 294.90 Wild resource (Wild
European Plum)
Mountains in Birsay, Xinyuan Country, Ili
27 YE-3 43.500 408 83.697 036 1 266.10 Wild resource (Wild
European Plum)
Mountains in Birsay, Xinyuan Country, Ili
28 YE-8 43.377 096 82.097 943 1 354.40 Wild resource (Wild
European Plum)
Mountains in Yiligedai, Gongliu Country, Ili
29 YE-9 43.377 708 82.097197 1 374.10 Wild resource (Wild
European Plum)
Mountains in Yiligedai, Gongliu Country, Ili
30 YE-10 43.377 738 82.097 034 1 377.10 Wild resource (Wild
European Plum)
Mountains in Yiligedai, Gongliu Country, Ili
449
Agricultural Science & Technology 2015
Table 2 Annealing temperatures and amplification results of primers
Serial No. Primer Sequence of primer
Recommended
annealing
temperature∥℃
Optimum
annealing
temperature∥℃
Number of
amplified
bands
Number of
polymorphic
bands
Ratio of
polymorphic
bands∥%
1 UBC811 GAGAGAGAGAGAGAGAC 52.00 54.00 15 13 86.67
2 UBC812 GAGAGAGAGAGAGAGAA 50.00 52.50 13 8 61.54
3 UBC816 CACACACACACACACAT 50.00 52.50 14 12 85.71
4 UBC817 CACACACACACACACAA 50.00 52.00 21 17 80.95
5 UBC826 ACACACACACACACACC 52.00 52.00 16 12 75.00
6 UBC827 ACACACACACACACACG 52.00 55.00 24 18 75.00
7 UBC836 AGAGAGAGAGAGAGAGYA 53.00 54.50 17 13 76.47
8 UBC841 GAGAGAGAGAGAGAGAYC 55.00 56.00 25 20 80.00
9 UBC844 CTCTCTCTCTCTCTCTRC 55.00 55.00 23 18 78.26
10 UBC845 CTCTCTCTCTCTCTCTRG 55.00 54.00 16 11 68.75
11 UBC857 ACACACACACACACACYG 55.00 53.00 29 24 82.76
12 UBC861 ACCACCACCACCACCACC 60.00 63.00 17 10 58.82
13 UBC866 CTCCTCCTCCTCCTCCTC 60.00 59.00 15 12 80.00
14 UBC873 GACAGACAGACAGACA 48.00 50.50 29 24 82.76
15 UBC888 BDBCACACACACACACA 51.33 54.00 23 19 82.61
16 UBC889 DBDACACACACACACAC 50.67 53.00 20 15 75.00
Total 317 246 77.60
Average 19.8 15.4 -
UBC840 respectively (Fig.1). As
shown in Fig.1, the bands amplified by
UBC836 were more than those ampli-
fied by UBC840. So UBC836 was se-
lected. Subsequently, the amplification
results of UBC836 were compared a-
mong different annealing tempera-
tures. As shown on electrophoresis,
the bands amplified at 54 and 55 ℃
were more and brighter than others. At
the same time, the two lanes had
cleaner backgrounds, and had no
nonspecific amplifications. Therefore,
54.5 ℃ was selected as the optimum
annealing temperature for UBC836.
The annealing temperatures of other
primers were determined as above.
Polymorphism analysis of primer
amplification
ISSR-PCR amplification of ge-
nomic DNAs of the 30 individual plants
of P. domestica in Xinjiang was per-
formed with 16 selected primers
(Table 2). A total of 317 bands, dis-
tributed between 150 to 2 600 bp,
were obtained from the amplification.
Among them, there were 246 poly-
morphic bands, accounting for
77.60%. It was suggested the genetic
diversity of P. domestica was abun-
dant in Xinjiang. The band numbers of
types differed among the 16 primers.
The numbers of bands amplified by
UBC857 and UBC873 were all 29,
ranking highest among the primers.
The number of bands amplified by
UBC812 was 13, ranking last. The av-
erage number of bands amplified by
the total 16 primers was 19.8. On the
other hand, the numbers of polymor-
phic bands amplified by UBC857 and
UBC873 were all 24, ranking first. But
the number of polymorphic bands am-
plified by UBC812 was only 8, ranking
last. The average number of polymor-
phic bands amplified by the total 16
primers was 15.4. The ratio of poly-
morphic bands for UBC811 was the
highest (86.67% ), while for UBC861
was the lowest (58.82%).
Genetic diversity analysis of P. do-
mestica individuals
The calculation results of genetic
diversity indexes of each amplification
site which came from the genomic
DNA of 30 tested individuals of P. do-
mestica in Xinjiang showed that the
average observed number of alleles
(Na) was 1.776 0; the average ef-
fective number of alleles (Ne) was
1.459 5; the average Nei’s gene diver-
sity index (H) was 0.266 6; the aver-
age Shannon’s information index (I)
was 0.399 1. The results above sug-
gested that there was a certain genetic
diversity among the 30 tested individu-
als (Fig.2). When the 30 individuals
were divided into 5 germplasm types
of P. domestica, the total gene diversi-
ty of the 30 individual plants (Ht) was
0.255 3; the average gene diversity
within the 5 germplasm types of P. do-
mestica (Hs) was 0.090 1; the Nei’s
P indicates primers; T indicates annealing temperatures,℃.
Fig.1 Agarose gel electrophoresis of PCR products with UBC836 and UBC840 at different
annealing temperatures
450
Agricultural Science & Technology2015
genetic differentiation coefficient (Gst)
was 0.647 1; the gene flow among dif-
ferent germplasm types (Nm) was
0.272 7. The results above showed
that there was 64.71% of genetic vari-
ation existing among the 5 different
germplasm types of P. domestica, and
there was only 35.29% of genetic vari-
ation existing within the 5 germplasm
types. So it could be concluded that
the genetic variation of P. domestica in
Xinjiang mainly exists among different
germplasm types, and the genetic dif-
ferentiation among different germ-
plasm types of P. domestica is greater
than that within germplasm types. The
gene flow among different germplasm
types was only 0.272 7 that was less
than 1, indicating that the gene flow
among the 5 germplasm types of
P. domestica was not high enough to
resist the genetic drift among different
germplasm types which resulted in the
genetic differentiation.
Similarity analysis of P. domestica
individuals
The 317 loci obtained from the
amplification of 30 individual plants of
P. domestica in Xinjiang by 16 primers
were used to calculate the 435 genetic
similarity coefficients between each
two individuals by software. The ge-
netic similarity coefficient between JS-
13 and JS-15, as well as between YE-
1 and YE-2, was the maximum. They
were all up to 1.000 0. So the four in-
dividuals were probably two pairs of
materials which were collected repeat-
edly. In addition, the genetic similarity
coefficients among JS-12, JS-13 and
JS-15, among YE-1, YE-8 and YE-9
and among YE-2, YE-8 and YE-9 all
reached 0.996 8, ranking second.
These materials were the most similar
individuals except the same individu-
als. However, the genetic similarity
coefficient between L1 and JS-6, as
well as between L1 and YE-3 and be-
tween L1 and YE-9, was only 0.555 2,
ranking last. It was also suggested that
the similarities among those materials
were the lowest.
Cluster analysis of P. domestica in-
dividuals
The cluster analysis of genetic
similarity coefficients was conducted
by unweighted pair-group method with
arithmetic means (UPGMA) (Fig.3).
Among the 5 germplasm types of
P. domestica in Xinjiang, all the tested
individuals of Wild European Plum, Ji-
ashi Smoked Plum and introduced
European plum varieties were clus-
tered in their own groups obviously.
But the individuals of Tacheng
Smoked Plum and Tacheng Binzi
were clustered in a same group with-
out obvious boundary. It was indicated
that Wild European Plum, Jiashi
Smoked Plum and introduced Euro-
pean plum varieties all have their own
special and obvious genetic charac-
teristics of germplasm types, respec-
tively. Although there were not genetic
characteristics that could be used to
distinguish between Tacheng Smoked
Plum and Tacheng Binzi, both of them
had common genetic characteristic
which could distinguish themselves
from the other three germplasm types.
The 30 tested individuals of
P .domestica were divided into 3 large
groups at about similarity coefficient of
0.719 (Line 1). The 4 introduced Euro-
pean plum varieties were clustered in-
to the first group; the second group
consisted of all the varieties of
Tacheng Smoked Plum and Tacheng
Binzi; the third group consisted of all
the varieties of Jiashi Smoked Plum
and Wild European Plum. In addition,
the third large group was further divid-
ed into two small groups, Jiashi
Smoked Plum and Wild European
Plum, at about similarity coefficient of
0.949 (Line 2). Above all, there was a
close genetic relationship between
Wild European Plum and Jiashi
Smoked Plum; and there were also
close genetic relationships among in-
troduced European plum varieties,
Tacheng Smoked Plum and Tacheng
Binzi. However, the similarity between
the first two germplasm types was
higher than those among the latter
Lanes indicate (from left to right) Marker, JS-1, JS-2, JS-3, JS-4, JS-5, JS-6, JS-11, JS-12,
JS-13, JS-14, JS-15, T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12 and YE-1, respec-
tively.
Fig.2 Agarose gel electrophoresis results of amplification products of UBC844 ISSR-PCR
of several individual plants
Fig.3 Cluster analysis of 30 tested individuals of P. domestica by UPGMA
451
Agricultural Science & Technology 2015
three types. In addition, all individuals
of Wild European Plum were classified
into one group at similarity coefficient
of 0.990; all individuals of Jiashi
Smoked Plum were classified into one
group at similarity coefficient of 0.983;
all individuals of introduced European
plum varieties were classified into one
group at similarity coefficient of 0.836;
all individuals of Tacheng Smoked
Plum and Tacheng Binzi were classi-
fied into one group just at similarity co-
efficient of 0.757. The differences in
similarity coefficient above reflected
that the genetic diversity of Wild Euro-
pean Plum was lowest among the 5
germplasm types of P. domestica in
Xinjiang. The genetic diversity of Jiashi
Smoked Plum was slightly higher than
that of Wild European Plum. More-
over, the genetic diversity of intro-
duced European plum varieties was
much higher than those of both of the
germplasm types above. The genetic
diversities of Tacheng Binzi and
Tacheng Smoked Plum were most
abundant.
Discussion
Genetic characteristics of germ-
plasm resources reflected by diver-
sity and similarity
The study results showed that the
genetic variation among different
germplasm types of P. domestica in
Xinjiang was much higher than that
within germplasm types, which was
similar or mostly close to the study re-
sults of Feng [11] on a few cultivars of
P. domestica and of Geng[24] on Wild
European Plum. It is indicated that the
genetic variation within germplasm
types is limited, and each of the
germplasm types all has its own
unique genetic characteristics. ISSR
markers can be applied to distinguish
different types of P. domestica
germplasm resources. In addition, the
gene flow among different types of
germplasm was only 0.272 7, indicat-
ing that there is a certain intension of
genetic drift existing within germplasm
types [25]. What’s more, the 5 germ-
plasm types of P. domestica were lack-
ing in gene flow, which was mainly re-
sulted from the long distances among
major germplasm types[2]. It is also re-
flected that people has done litter on
exploiting germplasm resources of
P. domestica. In addition to the 4 ma-
terials in pairs, there were all 3 plants,
among which the similarity coefficients
were up to 0.996 8, in each of the Wild
European Plum group and the Jiashi
Smoked Plum group. Compared with
other germplasm resources of P. do-
mestica, Wild European Plum and Ji-
ashi Smoked Plum showed a much
higher similarity within groups. It was
also showed that the two types of
germplasm resources have more sta-
ble genetic characteristic, as well as
stronger genetic drift and greater
chances of inbreeding. However, the
similarity coefficient between the in-
troduced European plum varieties and
the individuals of Jiashi Smoked Plum
was only 0.555 2. Despite the same
characteristics shared by the germ-
plasm types, it could be deduced that
there are big differences in genetic
characteristics between introduced
European plum varieties and Jiashi
Smoked Plum. Similarly, there are also
big differences in genetic characteris-
tics between introduced European
plum varieties and Wild European
Plum. Moreover, the previous study
on the appearance characteristics of
P. domestica also showed the similar
results.
Discussion of genetic relationship
and evolution based on cluster re-
sult
In the clustering, the individuals of
Wild European Plum grew in Birsay,
Xinyuan and grew in Yiligedai, Gongliu
were distributed in two different small
groups alternately, which were all in-
cluded in the large group of Wild Eu-
ropean Plum. It was suggested that
there might be certain varieties of Wild
European Plum, at the beginning of
their formation, existing in both of the
two places simultaneously. In other
words, the genetic relationship be-
tween the Wild European Plum grew in
Birsay, Xinyuan and those grew in
Yiligedai, Gongliu is very close. The
clustering results indicated that Jiashi
Smoked Plum had the closest rela-
tionship with Wild European Plum in
Xinjiang. Lin et al. [26] thought that Wild
European Plum is the ancestor of cul-
tivated European Plum. So the Jiashi
Smoked Plum might be the cultivated
type which is the closest one to the o-
riginal type. In addition, it was also indi-
cated that Tacheng Smoked Plum and
Tacheng Binzi were difficult to divide,
indicating similar, complicated and
abundant relationships within P. do-
mestica resources in Tacheng.
The research on parts of intro-
duced European plum varieties con-
ducted by Chen et al. [27] showed that
introduced varieties of plum were likely
to contain the gene of P. salicina. In
this study, the clustering results
showed that the introduced European
plum varieties had a closer genetic re-
lationship with Tacheng Smoked Plum
and Tacheng Binzi than with the other
local P. domestica germplasm types.
Therefore, it could be speculated that
there are certain P. domestica vari-
eties derived from the hybridization
between Tacheng Smoked Plum and
Tacheng Binzi and introduced Euro-
pean plum varieties. Although there is
almost no difference in appearance
between Tacheng Smoked Plum plant-
ed in Jiashi and Jiashi Smoked Plum,
all the individuals of Tacheng Smoked
Plum, except the individuals of
Tacheng Binzi, were divided into one
group according to cluster analysis.
This not only proved the ability of ISSR
markers to identify the essence of dif-
ferent germplasm types of P. domesti-
ca, but also showed the relatively
strong heritability of Tacheng Smoked
Plum[25]. Moreover, it was also indicat-
ed that there is no significant correla-
tion between the genetic relationships
among samples and distances among
cultivated locations, which is same
with the research results of Song et
al.[28] on P. cerasifera.
There are studies showing that in
the clustering, the greater the variation
range of similarity coefficient becomes,
the more abundant the genetic diver-
sity of the germplasm type is, and the
more complicated the genetic back-
ground of the germplasm type is[29-30].
In this research, the genetic diversity
of Wild European Plum was the low-
est, followed by Jiashi Smoked Plum.
However, the genetic diversity of the
Tacheng Smoked Plum and Tacheng
Binzi was most abundant, followed by
introduced European plum varieties. In
other words, the genetic backgrounds
of Wild European Plum and Jiashi
Smoked Plum are simple, so they are
452
Agricultural Science & Technology2015
both simplex germplasm types. How-
ever, the genetic backgrounds of the
Tacheng Smoked Plum, Tacheng
Binzi and the introduced European
plum varieties are complex, so they
are all genetic variation-abundant
germplasm types. In conclusion, the
evolutions of introduced European
plum varieties and P. domestica
germplasm resources in Tacheng are
more complex than those of Wild Eu-
ropean Plum and Jiashi Smoked
Plum.
Conclusions
The results of ISSR markers on
30 individuals of 5 types of P. domes-
tica germplasm resources in Xinjiang
showed that 317 bands were amplified
by 16 primers in total. The polymorphic
percentage of bands reached 77.60%.
There is a certain genetic diversity
among all the tested P. domestica
germplasm resources in Xinjiang. And
the genetic variation among different
germplasm types of P. domestica in
Xinjiang is much greater than that
within germplasm types. The cluster
analysis indicated that the tested indi-
viduals of P. domestica were divided
into 4 groups in total at similarity coef-
ficients of 0.719 and 0.949. The 4
groups included the group of intro-
duced European plum varieties, the
group of Tacheng Smoked Plum and
Tacheng Binzi, the group of Jiashi
Smoked Plum and the group of Wild
European Plum. Among all the groups,
the group of Wild European Plum had
the lowest genetic diversity, while the
group of Tacheng Smoked Plum and
Tacheng Binzi had the most abundant
genetic diversity. There is a very close
genetic relationship between Jiashi
Smoked Plum and Wild European
Plum. In addition, there are also close
genetic relationships among intro-
duced European plum varieties,
Tacheng Smoked Plum and Tacheng
Binzi.
References
[1] ZHANG JY (张加延), ZHOU E (周恩).
China’s Fruit Trees: Plum(中国果树志·
李卷)[M]. Beijing: China Forestry Press
(北京: 中国林业出版社), 1997.
[2] LIAO K(廖康). Study on wild fruit tree re-
sources in Xinjiang (新疆野生果树资源
研究)[M]. Urumqi: Xinjiang Science and
Technology Press (新疆: 新疆科技出版
社), 2013.
[3] LIN PJ(林培钧), LIAO MK(廖明康), SHI
L(施丽), et al. Finding and distribution of
wild Prunus domestica L (P. communis
Fritsch) in Yili, Xinjiang(first report)(新疆
伊犁野生欧洲李 Prunus domestica L
(P. communis Fritsch) 的发现与分布(第
一报))[J]. Liaoning Fruit Trees (北方果
树), 1986, 1: 1-3.
[4] WANG L (王磊 ). Wild Hawthorn, Wild
European Plum, Wild Rose and other
wild resources of fruit trees in Xinjiang
(新疆野山楂、野欧洲李、野蔷薇等野生
果树资源 ) [J]. Xinjiang Agricultural Sci-
ences(新疆农业科学), 1990, 3: 78-79.
[5] LIU WS(刘威生). Studies on the genetic
diversity among plum germplasm re-
sources and the phylogenetic relation-
ship of main plum species (李种质资源
遗传多样性及主要种间亲缘关系的研
究 ) [D]. Beijing: China Agricultural Uni-
versity(北京: 中国农业大学), 2005.
[6] WANG YZ (王玉柱 ), YANG L (杨丽 ),
YAN AL (阎爱玲 ), et al. Progress of
plum breeding in the world (李品种选育
研究进展 ) [J]. Journal of Fruit Science
(果树学报), 2002, 19(5): 243-244.
[7] ZOHARY D. Is the European plum,
Prunus domestica L, a P cerasifera
EHRH × P spinosa L allopolyploid [J].
Euphytica, 1992, 60: 75-77.
[8] ERYOMINE GV. New data on origin of
Prunus domestica L. [J]. Acta Horticul-
turae, 1991, 283: 27-29.
[9] HOU B(侯博), XU Z(许正). Study on wild
fruit trees and their kindred species in
the Yili Valley, China(中国伊犁野生果树
及近缘种研究 )[J]. Arid Zone Research
(干旱区研究), 2006, 23(3): 455.
[10] GENG WJ (耿文娟 ), LIAO K (廖康 ),
DIAO YQ(刁永强), et al. SSR analysis
on Wild European Plum genetic rela-
tionship of different populations(野生欧
洲李不同居群亲缘关系的 SSR 分析 )
[J]. Acta Horticulturae Sinica (园艺学
报), 2012, 39(supplement): 2602.
[11] FENG CJ (冯晨静). Phylogenetic rela-
tionships and genetic diversity re-
vealed by RAPD, SSR, ISSR markers
in Prunus (李种质资源 RAPD、SSR、
ISSR 亲缘关系鉴定及遗传多样性研
究 ) [D]. Baoding: Hebei Agricultural
University(保定: 河北农业大学), 2005.
[12] LIANG M(梁敏 ), DONG WX(董文轩 ),
ZHEN R(甄睿), et al. ISSR analysis of
genetic relationship of Peach, Plum
and Apricot resources (桃李杏资源亲
缘 关 系 的 ISSR 分 析 ) [J]. Guizhou
Agricultural Sciences (贵州农业科学),
2011, 39(5): 27-31.
[13] DECROOCQ V, HAGEN LS, FAV魪 M
G, et al. Microsatellite markers in the
hexaploid Prunus domestica species
and parentage lineage of three Euro-
pean plum cultivars using nuclear and
chloroplast simple-sequence repeats
[J]. Molecular breeding, 2004, 13:
135-138.
[14] SHIMADA T, HAYAMA H, HAJI T, et
al. Genetic diversity of plums charac-
terized by random amplified polymor-
phic DNA(RAPD) analysis[J]. Euphyti-
ca, 1999, 109: 143.
[15] ZHANG QL(张青林), LUO ZR(罗正荣).
ISSR technology and its applications
in fruit trees (ISSR 及其在果树上的应
用)[J]. Journal of Fruit Science(果树学
报), 2004, 21(1): 54-58.
[16] WANG XL(王小利), ZHANG GS(张改
生 ), LI HX (李红霞 ). Plant molecular
and cytogenetic experiment (植物分子
细 胞 遗 传 学 实 验 ) [M]. Shanghai:
Shanghai Science and Technology
Press(上海科学技术出版社), 2010.
[17] BAI JJ (白锦军 ), WEI AZ (魏安智 ),
WANG J (王佳), et al. Optimization of
an ISSR analysis system of Apricot of
kernel-consuming based on orthogo-
nal design(杏仁用 ISSR 分析体系的正
交优化)[J]. Molecular Plant Breeding(分
子植物育种), 2009, 7(6): 1237-1224.
[18] WANG HN (王会娜 ). The researches
on blossom biology and the analysis
on genetic relationship using ISSR of
the series Prunus varieties of ‘Ziguiqi-
ulu’(秭归秋露李系列品种开花生物学
研 究 及 ISSR 分 析 ) [D]. Changsha:
Central South University of Forestry
and Technology (长沙: 中南林业科技
大学), 2012.
[19] HE JW (何建文), YANG WP (杨文鹏),
HAN SY (韩世玉 ), et al. Single major
influencing factor and it is orthogonal
optimized design of SRAP-PCR in Hot
Pepper (Capsicum annum L.) ( 辣 椒
SRAP-PCR 反应体系主要影响因素的
单因素和正交设计优化)[J]. Seed (种
子), 2009, 28(9): 25-27.
[20] WANG J(王进), HE Q(何桥), OU Y(欧
毅 ), et al. Germplasm identification
and phylogenetic analysis of Plum us-
ing ISSR marker(李种质资源 ISSR 鉴
定及亲缘关系分析)[J]. Journal of Fruit
Science(果树学报), 2008, 25(2): 182-
187.
[21] QIAO YS (乔玉山 ). Establishment of
RAPD, ISSR and SSR reaction sys-
tem and analysis of genetic diversity of
Japanese Plum cultivars ( 中 国 李
RAPD、ISSR 和 SSR 反应体系的建立
及其品种资源遗传多样性分析 ) [D].
Nanjing: Nanjing Agricultural Universi-
ty(南京: 南京农业大学), 2003.
[22] GOUL魨O L, CORVO LM, OLIVEIRA
CM. Phenetic characterization of Plum
cultivars by high multiplex ratio mark-
ers: amplified fragment length poly-
morphisms and inter-simple sequence
repeats[J]. J Amer Soc Hort Sci, 2001,
126(1): 72-74.
[23] Y魱LMAZ KU, ERC魱CL魱 S, ASMA BM, et
al. Genetic relatedness in Prunus
genus revealed by inter-simple se-
quence repeat markers [J]. Hort Sci-
(Continued on page 503)
453
Agricultural Science & Technology2015
!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
(Continued from page 453)
Responsible editor: Tingting XU Responsible proofreader: Xiaoyan WU
ence, 2009, 42(2): 293-295.
[24] GENG WJ (耿 文 娟 ). Research on
germplasm resources characteristic
and genetic relationship for Wild Eu-
ropean Plum(Prunus domestica L.)(野
生欧洲李种质资源特性及亲缘关系研
究)[D]. Urumqi: Xinjiang Agricultural U-
niversity (乌鲁木齐 : 新疆农业大学 ),
2011.
[25] LI WJ (李惟基). Genetics (遗传学)[M].
Beijing: China Agricultural University
Press (北京 : 中国农业大学出版社 ),
2007.
[26] LIN PJ(林培钧), CUI NR(崔乃然). Wild
fruit forests resources in Tianshan
Mountains: Comprehensive research
on wild fruit forests in Ili, Xinjiang, Chi-
na(天山野果林资源——伊犁野果林综
合 研 究 ) [M]. Beijing: China Forestry
Publishing House(北京: 中国林业出版
社), 2000.
[27] CHEN H (陈红), YANG YR (杨迤然).
Genetic diversity and relationship of re-
sources in Guizhou analyzed by ISSR
markers (贵州李资源遗传多样性及亲
缘关系的 ISSR 分析 ) [J]. Journal of
Fruit Science (果树学报), 2014, 31(2):
179.
[28] SONG CM(宋常美), WEN XP(文晓鹏),
YANG ET(杨尔泰). Cherry germplasm
from Guizhou Province analyzed by
ISSR markers(贵州樱桃李种质资源的
ISSR 分析)[J]. Acta Horticulturae Sini-
ca(园艺学报), 2011, 38(8): 1535.
[29] ZHANG BX(张冰雪), FAN FH(范付华),
QIAO G (乔光), et al. DNA fingerprint
construction and genetic relationship
analysis of dragon fruit germplasm in
Guizhou by ISSR markers(贵州地方火
龙果芽变种质 DNA 指纹图谱及遗传多
样性的 ISSR 分析)[J]. Journal of Fruit
Science(果树学报), 2013, 30(4): 577.
[30] LI JL (李俊丽 ), XIANG CP (向长萍 ),
ZHANG HR(张宏荣), et al. Analysis of
genetic diversity in Cucurbita by RAPD
markers (南瓜种质资源遗传多样性的
RAPD 分 析 ) [J]. Acta Horticulturae
Sinica (园艺学报), 2005, 32 (5): 838-
839.
Responsible editor: Xiaohui FAN Responsible proofreader: Xiaoyan WU
dation of membrane lipid in hybrid rice
seedlings under osmotic stress (维生素
C 对渗透胁迫下杂交稻幼苗膜脂过氧化
作用的影响)[J]. Hybrid Rice(杂交水稻),
2003, 18(3): 59-61.
[6] YANG P (杨萍 ), LUO QX (罗庆熙 ),
ZHANG KK (张珂珂). Effects of exoge-
nous vitamins on seed germination of
cucumber under salt stress(外源维生素
对盐胁迫下黄瓜种子发芽特性的影响)
[J]. Journal of Southwest China Normal
University: Natural Science Edition (西
南师范大学学报: 自然科学版), 2010(3):
109-111.
[7] ZHANG HT (张洪涛), AN WS (安旺盛),
ZENG FL (曾福礼). Effects of vitamin C
on peroxidization of chloroplast mem-
brane lipid from cucumber(维生素 C 对
黄瓜叶绿体膜脂过氧化的影响)[J]. Acta
Botanica Boreali-Occidentalia Sinica(西
北植物学报), 1999, 19(2): 321-324.
[8] WANG W (王薇), WANG YJ (王亚君),
CHEN ZG(陈志刚). Effects of vitamin C
on seed germination of muskmelon un-
der salt stress (维生素 C 浸种对盐胁迫
下甜瓜种子萌发的影响 ) [J]. Jilin Veg-
etable(吉林蔬菜), 2013(3): 33-34.
[9] TANG ZJ(唐祖军), SONG M(宋明). Ef-
fects of PEG, CaCl2 and vitamin C on
seed vigor of Chinese cabbage (Brassi-
ca campestris L. ssp. pekinensis)(PEG、
CaCl2和维生素 C 对大白菜种子活力的
影响)[J]. Journal of Southwest Agricul-
tural University (西南农业大学学报 ),
1999, 21(5): 430-432.
[10] XU ZJ(徐振军), LI WH(李文辉), BAO N
(包楠). Effects of vitamin C soaking on
germination of alfalfa seeds under dif-
ferent salinity stress (维生素 C 浸种对
盐胁迫下紫花苜蓿种子发芽特性的影
响)[J]. Seed (种子), 2009, 28 (7): 42-
44.
[11] YU ZX(于志贤), GENG K(耿稞), HOU
JH (侯建华), et al. Effect of salt stress
on seed germination of different sun-
flower (盐胁迫对不同基因型向日葵种
子萌发的影响)[J]. Seed (种子), 2013,
32(10): 28-33.
[12] ZHANG LJ(张立军), FAN JJ(樊金娟).
Experimental Course of Plant Physiol-
ogy(植物生理学实验教程)[M]. Beijing:
Higher Education Press (北京: 高等教
育出版社), 2006: 92-103.
[13] LIU HL(刘洪兰), LI JF(李景富), XU XY
(许向阳 ), et al. Influence of salt con-
centration on germination of different
tomato seeds(NaCl 胁迫对不同番茄种
子萌芽的影响)[J]. Journal of Northeast
Agricultural University (东北农业大学
学报), 2008, 39(5): 28-33.
[14] YANG JW(杨建伟), LIU Z(刘征), DU L
(杜丽 ), et al. Effects of salt stress on
seed germination and seedling growth
of Capsicum annuum(盐胁迫对三樱椒
种 子 萌 发 及 幼 苗 生 长 的 影 响 ) [J].
Jiangsu Agricultural Sciences (江苏农
业科学), 2012, 40(2): 120-122.
[15] REN YF(任艳芳), HE JY(何俊瑜), HE
SJ (何师加 ). Effects of salt stress on
germination of lettuce seed and growth
of seedling (盐胁迫对莴苣种子萌发和
幼苗生长的影响)[J]. Northern Horticul-
ture(北方园艺), 2008, (8): 35-36.
[16] LI CZ (李存桢 ), LIU XJ (刘小京 ),
HUANG W (黄玮), et al. Effect of salt
stress on seed germination and its re-
covery of Atriplex centralasiatica(不同
盐分胁迫对中亚滨藜种子萌发及其恢
复的影响 ) [J]. Journal of Agricultural
University of Hebei (河北农业大学学
报), 2005, 28(6): 1-3, 14.
[17] YAO QL (姚启伦), ZHANG W (张维).
Effects of NaCl-stress on seed germi-
nation and plant morphology of corn in-
bred lines (NaCl 胁迫对玉米自交系种
子萌发和幼苗生长的影响 )[J]. Journal
of Anhui Agricultural Sciences (安徽农
业科学), 2010, 38(5): 2294-2296.
[18] WANG JC (王冀川), XU YL (徐雅丽),
JIANG L ( 姜 莉 ). Research on salt
stress affects on seed vigor and phys-
iological and biochemical of oil-sun-
flower seeding(盐胁迫对油葵种子活力
和幼苗生理生化特性的影响)[J]. Seed
(种子), 2004, 23(5): 18-20.
[19] WANG JH (王建华), LIU HX (刘鸿先),
XU T (徐同 ). The role of superoxide
dismutase (SOD) in stress physiology
and senescence physiology of plant(超
氧化物歧化酶 SOD 在植物逆境和衰老
生理中的作用 ) [J]. Plant Physiology
Communications (植物生理学通讯 ),
1987(1): 1-7.
503