全 文 ：书Optimization of ISSR-PCR Reaction System and
Preliminary Construction of ISSR Fingerprinting of
Some Species in Bryaceae
WANG Chen-ying1* ，ZHAO Jian-cheng2
1． Department of Life Science，Zhengzhou Teachers College，Zhengzhou 450044; 2． College of Life Science，Hebei Normal University，Shijiazhuang
050016
Abstract ［Objective］The aim was to provide molecular basis for the identification of species in the moss family Bryaceae by the construction of
inter-simple sequence repeats ( ISSR) fingerprinting． ［Method］ In order to seek standardizing PCR reaction set-up，an orthogonal design was
used to optimize ISSR-PCR amplification system of Bryaceae in five factors ( Mg2 +，dNTPs，primer，DNA template，Taq DNA polymerase) at
four levels respectively． ［Result］A suitable ISSR reaction system was obtained，namely: 20 μl reaction system containing 5 ng of DNA tem-
plate，0． 2 μmol /L primer，2． 25 mmol /L MgCl2，0． 6 U of Taq DNA polymerase，0． 4 mmol /L dNTPs． Proper annealing temperature was found at
48 －50 ℃． The above system and six ISSR-PCR primers were used for the PCR amplification of 14 samples from Bryaceae and the related spe-
cies in Mniaceae． A total of 86 bands were amplified，all showed polymorphism． NJ cluster analysis showed a star-shaped cladogram． ［Conclu-
sion］The results manifested that ISSR fingerprinting could provide the appropriate degree of polymorphism at low taxonomic level，so it would be
a useful tool to provide additional evidence for resolving taxonomic relationships at the species level of Bryaceae．
Key words Bryaceae; ISSR; Optimization of reaction system; Species-level; Taxonomic relationship
Received: July 28，2011 Accepted: October 10，2011
Supported by Natural Science Foundation of Hebei Province
( C2006000147 ) ; Zhengzhou Science and Technology Program
( 10PTGN449-6) ．
* Corresponding author． E-mail: ld1988jwx@tom． com
Bryaceae is a large family in moss． Traditionally，its clas-
sification system is primarily established on the hypotheses or
assumptions based on the comparable morphology character-
istics ( such as the sporophyte or gametophyte) ． Due to the
original hypotheses used，various researches have provided
different solutions，thus leading to the confusion on the identi-
fication of Bryaceae species． In addition，some critical mor-
phological characteristics do not always play their role in the i-
dentification of Bryaceae plants at the species level，not to
mention the morphological characteristics influenced and
changed by the environment factors． Therefore，its quite diffi-
cult to successfully classify the Bryaceae species［1］． In this
case，it is necessary to develop more precise techniques such
as molecular markers to provide a high degree of polymor-
phism and the different characteristics for the classification of
Bryaceae plants．
Zietkiewicz et al．［2］ clarified the inter-simple sequence re-
peats ( ISSR) method in 1994． With the anchored simple se-
quence repeats ( SSR) as primers，ISSR amplifies a DNA
fragment defined by two opposite microsatellite or SSR． Be-
cause the evolutionary rate of microsatellite DNA is higher
than that of other types of DNA，ISSR markers will be more
probably to provide polymorphism than most of other types of
DNA ( including RAPD) ． Meanwhile，ISSR has longer primers
sequences and higher annealing temperature than the RAPD
technique． QIAN Wei et al．［3］ suggested that ISSR would pro-
duce more reliable and reproducible bands． In addition，this
method is simple and lower cost，and can generate many
markers with unknown DNA sequence information［4］． Studies
have shown that ISSR is an effective tool in the classification
of plants at species level［5］ However，this method has not yet
applied in the classification of Bryaceae plants．
In this study，ISSR method was used to find some char-
acteristics which could distinguish closely related species
directly at DNA level so as to provide basic information for the
further resolving of taxonomic relationships of Bryaceae at the
species level．
Materials and Methods
Classification sampling
Based on Flora Bryophytorum Sinicorum［6］，14 spe-
cies of Bryaceae plants and related families and genera were
selected as materials． Voucher specimens information was lis-
ted in Table 1，and the voucher specimens were deposited in
Hebei Normal University Herbarium ( HBNU) ．
DNA extraction
Samples were packed in paper bag after collection，after
air dried，they were stored at － 80 ℃． The green parts of
plants were cut and placed in Eppendorf centrifuge tube，and
then the DNA extraction buffer was added． After grinding with
an electric drill，different types of DNA samples were extrac-
ted using DNA extraction kit ( Tiangen Biotech ( Beijing) Co．，
Ltd． ) ． The quality and concentration of the extracted DNA
samples were detected by NanoDropTM 1000 UV/visible spec-
trophotometer ( Thermo Scientific) ．
Establishment of PCR reaction system ( pre-test)
The DNA samples of B． argenteum leaf were used as
template to establish the initial ISSR-PCR reaction system，
PCR amplification procedures and ISSR primers based on the
method of Hassel et al．［4］( Table 2) ．
The 20 μl PCR reaction system included 2 μl of 10 × Taq
Plus buffer ［200 mmol /L Tris-HCl ( pH 8． 4 ) ，200 mmol /L
KCl，100 mmol /L ( NH4 ) 2SO4，15 mmol /L MgCl2，etc．］，10
ng DNA template，10 μl of 10 μmol /L primers，0． 60 μl of 25
mmol /L Mg2 +，0． 12 μl of 5 U/μl Taq plus DNA polymerase
Agricultural Science ＆ Technology，2011，12( 11) : 1561 －1564，1568
Copyright 2011，Information Institute of HAAS． All rights reserved． Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2011.11.001
( Tiangen Biotech ( Beijing) Co．，Ltd． ) ，0．4 μl of 10 mmol /L
dNTPs，and the water was added to take the total system to
20 μl． One replication was performed． The solutions without
DNA samples were considered as the control． PCR reaction
was carried out on 5331 Mastercycler gradient PCR instrument
( Eppendorf，Germany) with the reaction conditions as: 94 ℃
denaturation for 4 min; 35 cycles of 94 ℃ denaturation for 1
min，48 －50 ℃ ( for different primers) annealing for 2 min and
72 ℃ extension for 1 min; 72 ℃ extension for 7 min，then
stored at 4 ℃． PCR products were separated on 2% ( W/V)
agarose gel containing Goldview ( Beijing SBS Genetech Co．，
Ltd． ) nucleic acid dye with the voltage of 4．3 V/cm． After the e-
lectrophoresis，the gel was placed in GeneGenius Super12 auto-
matic image analysis system for observing and photographing．
Table 1 Voucher specimen information
Family Genus Species Herbarium voucher
Bryaceae Anomobryum Anomobryum gemmigerum Broth Laowo of Lushui County in Yunnan Province，20073730． L． B． Li
Brachymenium Brachymenium longicolle Thér． Laowo of Lushui County in Yunnan Province，20073687． L． B． Li
Bryum Bryum argenteum Hedw． Majiazi of Pingquan County in Hebei Province，20050031． J． C． Zhao
Bryum pallescens Schleich． Longmudong of Liaoheyuan natural reverse in Hebei Province，
ex Schwaegr． 20050088． D． Wang
Rhodobryum Rhodobryum giganteum Labahe natural reverse of Quanxian County in Sichuan Province，
( Schwaegr． ) Par． 20070472． J． C． Zhao
Plagiobryum Plagiobryum zierii ( Hedw． ) Lindb． Maojingba of Longhua County in Hebei Province，000514． W． Q． Li
Pohlia Pohlia annotina ( Hedw． ) Lindb． Hiroshima greening center of Japan，J004． J． C． Zhao
Pohlia elongata Hedw． Labahe natural reverse of Quanxian County in Sichuan Province，
20070940． J． C． Zhao
Pohlia crudoides ( Sull． ＆ Lesq． )
Broth． Urumqi Nanshan Mountain Forest Farm in Xinjiang，
070809． J． C． Zhao
Pohlia nutans ( Hedw． ) Lindb． Hiroshima greening center of Japan，J013． J． C． Zhao
Pohlia proligera ( Kindb． ex Limpr． ) Hiroshima greening center of Japan，J008． J． C． Zhao
Lindb． ex Arn．
Pohlia longicollis ( Hedw． ) Lindb． Kowloon waterfall of the Baiyun Montain in Songxian County of
Henan Province，97067． Y． Z． Ye
Mniaceae Mnium Mnium hornum Hedw． Shaoshan of Xiangtan in Hunan Province，20080015． J． C． Zhao
Plagiomnium Plagiomnium cuspidatum Tuoliang of Pingshan County in Hebei Province，
( Hedw． ) T． Kop． 20070925． C． Y． Wang
Table 2 ISSR primer sequences ( from the University of British
Columbia) used for Bryum argenteum，with primer specif-
ic annealing temperatures
Primer Primer sequence( 5 －3) Annealingtemperature∥℃
808 AGA GAG AGA GAG AGA GC 50
811 GAG AGA GAG AGA GAG AC 50
812 GAG AGA GAG AGA GAG AA 50
818 CAC ACA CAC ACA CAC AG 50
825 ACA CAC ACA CAC ACA CT 48
826 ACA CAC ACA CAC ACA CC 48
Optimization of ISSR-PCR reaction system
In order to obtain the bands with clear boundaries，clear
background，good repeatability and proper brightness，the
primer UBC808 was used to further optimize the PCR reaction
system． PCR annealing temperature，substrate concentration
and other factors will affect the number and length of amplified
fragment of a specific primer，so the orthogonal experimental
design with five factors ( Mg2 +，dNTPs，primers，DNA tem-
plate，Taq DNA polymerase) and 4 levels ( Table 3) were uti-
lized to optimize the PCR amplification system． PCR reaction
conditions were the same with that of Establishment of PCR
reaction system ( pre-test) ，and the annealing temperature
was 50 ℃． The total number of combinations for orthogonal
experiments was 16． Each PCR reaction tube was added with
2 μl 10 × Taq Plus buffer，the corresponding reagents and
DNA templates，then the water was added till 20 μl． In addi-
tion，the replications were set．
ISSR polymorphism of mosses samples
With six selected ISSR-PCR primers and the optimized
standard ISSR reaction system，14 samples from Bryaceae
and Mniaceae were amplified． The ISSR-PCR amplification
bands of each sample were recorded as 1 for with bands
and 0 for without bands． Based on the repeatable and relia-
ble records，the original data matrix was obtained． Neighbor-
joining ( NJ) in PAUP Version 4． 0b10［7］ software was used
for cluster analysis．
Table 3 ISSR reaction system produced by means of orthogonal ex-
perimental design method L16 ( 4
5 )
No． Mg
2 +
mmol /L
dNTPs
mmol /L
Primer
μmol /L
DNA
Template
ng /20 μl
Taq DNA
polymerase
U/20 μl
1 1． 75 0． 1 0． 1 5 0． 4
2 2． 00 0． 2 0． 2 10 0． 4
3 2． 25 0． 3 0． 3 15 0． 4
4 2． 50 0． 4 0． 4 20 0． 4
5 1． 75 0． 2 0． 4 15 0． 6
6 2． 00 0． 1 0． 3 20 0． 6
7 2． 25 0． 4 0． 2 5 0． 6
8 2． 50 0． 3 0． 1 10 0． 6
9 1． 75 0． 3 0． 2 20 0． 8
10 2． 00 0． 4 0． 1 15 0． 8
11 2． 25 0． 1 0． 4 10 0． 8
12 2． 50 0． 2 0． 3 5 0． 8
13 1． 75 0． 4 0． 3 10 1． 0
14 2． 00 0． 3 0． 4 5 1． 0
15 2． 25 0． 2 0． 1 20 1． 0
16 2． 50 0． 1 0． 2 15 1． 0
Results and Analysis
Screening of the appropriate primers
Six primers mentioned by Hassel et al．［4］ could produce
bands in the experiment，however，the intensity and resolu-
tion of the bands was lower ( Fig． 1) ． Therefore，PCR reac-
tion system was needed to be further optimized．
2651 Agricultural Science ＆ Technology Vol． 12，No． 11，2011
M: Marker ( λDNA +BamHⅠ+HindⅢ) ; 2 －3: Primer 808; 4 －
5: Primer 811; 6 －7: Primer 812; 8 －9: Primer 818; 10 －11:
Primer 825; 12: Primer 826．
Fig． 1 Amplification results using different primers
Optimization of ISSR-PCR reaction system
In order to obtain the bands with clear boundaries，clear
background，good repeatability and proper brightness，the
primer UBC808 and the DNA samples of B． argenteum were
used for further PCR reaction system optimization． Fig． 2
showed that except for combination 1，6，8，9，10，11，15
and 16，the remaining combinations showed bands，in which
combination 7 showed the clearest bands ( 10 bands) ． There-
fore，the optimal PCR reaction system was: 20 μl reaction
mixture including 5 ng of DNA template，0．2 μmol /L primers，
2．25 mmol /L Mg2 +，0． 6 U of Taq DNA polymerase，0． 4
mmol /L dNTPs．
M: Marker ( λDNA +BamHⅠ+HindⅢ) ; 1 －2: Treatment 1; 3 －4: Treatment 2; 5 －6: Treatment 3; 7 －8: Treatment 4; 9 －10: Treat-
ment 5; 11 －12: Treatment 6; 13 －14: Treatment 7; 15: Treatment 8; 16: Negative control ( Treatment as shown in Table 3) ．
Fig． 2 Electrophoresis of ISSR-PCR orthogonal design
Lanes 1 －14 represent samples Anomobryum gemmigerum，Brachymenium longicolle，Rhodobryum giganteum，Plagiobryum zierii，Pohlia
elongata ，Plagiomnium cuspidatum，Bryum pallescens，Pohlia nutans，Pohlia annotina，Bryum argenteum，Pohlia proligera，Mnium hor-
num ，Pohlia crudoides ，Pohlia longicollis; Lanes 15 －16: Negative control ． M:Marker ( 100 bp DNA ladder) ．
Fig． 3 Electrophoresis map of ISSR amplification products
3651
WANG Chen-ying et al． Optimization of ISSR-PCR Reaction System and Preliminary Construction of ISSR Fingerprinting of Some
Species in Bryaceae
ISSR polymorphism
To ensure the repeatability of ISSR，the PCR amplifica-
tion was performed on each of the 14 samples using the six
ISSR primers and the optimized PCR reaction system ( Fig．
3) ． Six primers had amplified a total of 86 bands with a length
range of 200 －2 000 bp，and each primer generated an aver-
age of 14． 3 bands． There were 86 polymorphic bands，with
the polymorphic rate of 100%． The results showed that，ISSR
fingerprints could provide appropriate polymorphism at the
species level，and that the ISSR fingerprint built by primers
UBC-808，811 and 826 could distinguish all the tested plants．
Cluster analysis
According to the results of NJ cluster analysis，the ob-
tained cladogram was star-shaped ( Fig． 4 ) ． Among them，
P． annotina and P． longicollis clustered into the same branch，
which were significantly different from P． nutans; P． elongata
and M． hornum clustered into the same branch，while P． prol-
igera and P． cuspidatum clustered into one; P． crudoides and
A． gemmigerum clustered into one branch，which was sepa-
rated from other species．
Fig． 4 Unroeted neighbor-joining tree resulting from the analysis
of ISSR dataset
Discussion
Orthogonal design is an effective mathematical method
for multi-factor experimental design，and it can easily find the
best experimental scheme． In order to obtain stable and
repeatable amplification results，it is necessary to carry out
the ISSR-PCR reaction optimization． There are multiple fac-
tors affecting PCR amplification，such as Mg2 + concentration，
dNTPs concentration，Taq DNA polymerase concentration，
primer concentration and DNA template concentration and so
on． Generally，the orthogonal design is utilized to select the
optimal PCR reaction system and reduce experimental scale．
The results of L16 ( 4
5 ) orthogonal design showed that 2． 25
mmol /L Mg2 + was essential to obtain the bands with suitable
strength and clarity; 0． 03 U/μl Taq DNA polymerase could
ensure the clarity of the amplified bands; 0． 4 mmol /L dNTPs
could balance the contradiction between the decreased fidelity
of Taq DNA polymerase and the decreased PCR reaction effec-
tiveness; with the template concentration increasing，the band
intensity increased，but bands became diffuse，5 ng of DNA
template could ensure the intensity and identification of bands．
The use of small amounts of DNA for the analysis of moss
is meaningful． The size of many species of mosses resources，
especially the Bryaceae plants，is usually small，so the amount
of collected samples in field is limited． Therefore，this method is
useful to solve the problems on sample collection and destruc-
tion of habitat of moss，especially for some endangered spe-
cies．
Traditionally，plants of Pohlia are classified to Bryaceae
as its morphological characteristics［6，8 －10］． However，Cox et
al．［11 －13］ thought that the plants of Pohlia showed nearer rela-
tionship with the plants in Mniaceae according to the phyloge-
netic analysis based on DNA sequence of chloroplast ge-
nome，nuclear genome and mitochondrial genome． Crosby et
al．［14］ proposed that the Pohlia should be moved into Mniace-
ae． Therefore，the classification of plants in Pohlia had in-
duced a dispute． In this study，the higher level of polymor-
phism of ISSR markers provided a feasibility to distinguish the
closely related species of moss plants． Particularly，when the
morphological features were not enough to identify the species
of moss plants，DNA fingerprinting built by UBC-808，811，
826 primers could further provide additional information，which
is undoubtedly helped to solve the classification of plants of
Pohlia．
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4651 Agricultural Science ＆ Technology Vol． 12，No． 11，2011
Fig． 3 Detection result of Thai Hom Mali Rice mixed with
pahtum rice
identifications by sense and boiling in water; in addition，it is
simple，sensitive and cost-saving，suitable to be used in rou-
tine tests．
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Responsible editor: YIN Qing-qing Responsible proofreader:
檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪檪
WU Xiao-yan
泰国茉莉香米纯度的 RAPD鉴定( 摘要)
徐 颖* ( 北仑出入境检验检疫局，浙江宁波 315800)
［目的］针对现有的泰国茉莉香米品种纯度检测方法的不足，建立特异、灵敏、准确、具有实用性的泰国茉莉香米品种纯度检测方法。
［方法］通过对影响 RAPD结果的模板 DNA、Mg2 +离子、随机引物、dNTPs和 Taq酶的浓度条件进行优化、以及随机引物的筛选，建立了泰国茉
莉香米法定品种 KDML105和 RD15的双引物协同 RAPD检测方法。
［结果］得到最适宜的 RAPD反应体系为: 反应体积25 μl，模板 DNA浓度4 －32 ng /μl、随机引物浓度200 μg /L、Mg2 +浓度2． 0 mmo1 /L、dNTPs
浓度 200 μmol /L 和 Taq酶 1． 0 U。通过两条 DNA分子标记的双隐性特征可以区分茉莉香米和非茉莉香米品种。
［结论］该技术可以有效地弥补了感官法和水煮法的不足，具有操作简单、快速灵敏、费用低等优点，适合在日常检验中推广使用。
关键词 RAPD; 泰国茉莉香米;纯度
作者简介 徐颖( 1978 － ) ，女，上海人，硕士，研究方向:粮食检验，E-mail: bl． xuy@ nbciq． gov． cn。* 通讯作者。
收稿日期 2011-08-24 修回日期
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2011-09-26
( From page 1564)
真藓科植物 ISSR-PCR反应体系的优化及 ISSR指纹图谱的初步构建( 摘要)
汪琛颖1* ，赵建成2 ( 1．郑州师范学院生命科学系，河南郑州 450044; 2．河北师范大学生命科学学院，河北石家庄 050016)
［目的］试图通过 ISSR指纹图谱的构建，为真藓科( Bryacae) 植物的种类鉴定提供分子分析数据。
［方法］为获得标准试验程序，首先利用正交试验设计的方法对真藓科植物的 ISSR-PCR反应的 5 因素( Mg2 +、dNTPs、引物、DNA 模板、Taq
DNA 聚合酶) 4 水平进行试验。
［结果］最适扩增条件是:在 20 μl PCR反应体系中，5 ng 模板 DNA，0． 2 μmol /L 引物，2． 25 mmol /L MgCl2，0． 6 U Taq DNA 聚合酶，0． 4
mmol /L dNTPs。最适退火温度为 48 ～50 ℃。利用此结论使用 6 条 ISSR引物对 14 种真藓科及相关的提灯藓科( Mniaceae) 植物分别进行
PCR扩增，共扩增出 86 条带，多态性带为 86条，多态率达 100%。根据扩增结果进行 NJ聚类分析，得到的支序图呈星状。
［结论］ISSR指纹能够在种级分类水平提供适度的多态性，利用引物 UBC-808、811以及 826构建的 ISSR指纹图谱能够区分所有供试植物，为
利用 ISSR指纹技术解决真藓科植物种级水平分类关系问题时提供了分子辅助证据的可行性。
关键词 真藓科; ISSR; 反应体系优化;种级水平;分类关系
基金项目 河北省自然科学基金资助项目( C2006000147) ; 郑州市科技计划资助项目( 10PTGN449-6) 。
作者简介 汪琛颖( 1968 － ) ，女，河南南阳人，副教授，博士，从事苔藓植物分子系统发生及分类学研究，E-mail: okwcy@ yahoo． com． cn。
收稿日期 2011-07-28 修回日期 2011-10-10
8651 Agricultural Science ＆ Technology Vol． 12，No． 11，2011