全 文 ：RAPD Analysis for Genetic Diversity of Nineteen
Common and Tartary Buckwheat Varieties
DENG Lin-qiong1，2，ZHANG Kui1，HUANG Kai-feng1，CHEN Qing-fu1*
1． Institute of Plant Genetics and Breeding，School of Life Science，Guizhou Normal University，Guiyang 550001; 2． Institute of Ecological
Construction of Karst Mountain Area，Bijie University，Bijie 551700
Abstract ［Objective］The study aimed to carry out RAPD analysis of genetic diversity of common and tartary buckwheat varieties． ［Method］
The genetic diversity of 19 common and tartary buckwheat varieties including the tested varieties in Guizhou region during 1999 －2010 and their
parents were studied using 7 primers by means of random amplified polymorphic DNA ( RAPD) ． ［Result］A total of 149 DNA bands were ob-
tained． In which，141 bands were polymorphic，accounting for 94． 89%． Polymorphism analysis and cluster analysis showed that all varieties had
their own special bands different from each other． The varieties native to Weining were close to each other，and other common buckwheat varie-
ties were obviously different from each other． The interspecific genetic variation was the greatest; the intraspecific genetic variation of common
buckwheat varieties was greater than that of tartary buckwheat varieties． ［Conclusion］The RAPD fingerprints of the 19 buckwheat varieties were
established in present study．
Key words Common buckwheat; Tartary buckwheat; RAPD; Genetic diversity
Received: January 21，2011 Accepted: February 18，2011
Supported by Special Project of Guizhou Animal and Plant Breeding
( QianNongYuZhuanZi［2010］023) ，Natural Science Foundation of
China ( 31060207 ) ， and Key Project of Guizhou Agriculture
( QianKeHe NY Zi［2010］3094) ．
* Corresponding author． E-mail: cqf1966@163． com
Buckwheat ( Fagopyrum Mill，Polygonaceae ) includes
two cultivated species，namely F． esculentum Moench ( com-
mon buckwheat ) and F． tataricum ( L． ) Gaertn． ( tartary
buckwheat) ［1 －7］． It has high nutritional and medicinal values．
The well-balanced amino acid composition of buckwheat is
very suitable for human nutrition［8］． Buckwheat can effectively
control diabetes，prevent sclerosis of blood vessels and hy-
pertension． It also has the ability to invigorate stomach and
promote digestion，and increase the bodys immunity to dis-
ease． In addition， it also can prevent cancer． Moreover，
buckwheat is also helpful to the growth and mental develop-
ment of children［2，8 －9］．
With the development of molecular biology in recent
years，RAPD analysis based on polymerase chain reaction
( PCR) which was developed in late 1990s has become a kind
of common molecular marker technology for detecting DNA
polymorphism［10］． RAPD analysis has many advantages inclu-
ding relatively simple and convenient operation，high sensitivi-
ty，and low cost; in addition，it can not be affected by the en-
vironment，quantitative traits and biological cycle; it can ob-
jectively show the DNA differences among the tested materi-
als; therefore，it has been widely used in all aspects of molec-
ular biology study［11 －12］． Currently，RAPD technology is also
widely used in investigation of genetic relationship［13 －17］ and
intraspecific diversity［18 －23］ of plant species．
At present，there are some reports of interspecific genetic
relationship by RAPD analysis of buckwheat in domestic and
international researches． WANG Li-hua et al．［14］ analyzed the
relationship among 9 species，1 variety and 2 subspecies of
wild buckwheat resources in Yunnan by RAPD technology;
Ren Cui-juan et al．［17］ reported the RAPD genetic relationship
among 11 species of buckwheat． Tan Ping et al．［20］，Fan
Dong-li et al．［21］，and Sharma et al．［22］ reported RAPD diver-
sity of cultivated buckwheat varieties． Kump et al．［23］ investi-
gated the genetic diversity of 40 cultivated and wild buckwheat
materials by RAPD technology． In present study，The RAPD
genetic diversity of 19 common and tartary buckwheat varie-
ties including tested varieties in Guizhou during 1999 － 2010
and their parents were studied，in order to provide some evi-
dences on variety identification and buckwheat breeding．
Materials and Methods
Materials
The name，species name，ploidy and symbol of the ma-
terials were shown in Table 1． All the materials were provided
by Institute of Plant Genetics and Breeding，School of Life
Sciences，Guizhou Normal University，and all tests were
completed at the institute．
Methods
Material culture Seeds of the tested materials were im-
mersed in water for 12 h at room temperature and cultured in
28 ℃ light incubator． After germination，they were cultivated
in pots till three-four leaf stage． Finally，DNA of the healthy
and young leaf was extracted．
DNA extraction DNA was extracted by 2 × CTAB method
as follows． 0． 3 g fresh and healthy buckwheat leaf was
weighed，fully ground into powder in liquid nitrogen，and put
into 1． 5 ml centrifuge tube． Then，750 μl CTAB extraction
buffer ( 100 mmol /L Tris-HCl，50 mmol /L EDTA，2% CTAB，
1% PVP) was added． After 40 min of water bath at 65 ℃，
750 μl of chloroform-isoamyl alcohol mixture ( 24∶1，V/V) was
added and mixed well． The mixture was centrifuged at 10 000
r /min for 10 min; the supernatant was collected into a clean
centrifuge tube and added with an equal volume of chloroform-
isoamyl alcohol mixture ( 24∶1，V/V) ． After well mixing，the
mixture was centrifuged at 10 000 r /min for 10 min． The su-
pernatant was collected and added with 2 volumes of frozen
ethanol，and then the tube was gently shaken until flocculent
precipitate could be seen． After the tube was placed in －20 ℃
for 30 min，the supernatant was discarded，and the precipi-
Agricultural Science ＆ Technology，2011，12( 1) : 65 －69
Copyright 2011，Information Institute of HAAS． All rights reserved． Agricultural Biotechnology
DOI:10.16175/j.cnki.1009-4229.2011.01.016
tate was washed with frozen 70% ethanol for 3 times and dried
at room temperature． Finally，TE buffer ( 10 mmol /L Tris-HCl，
1 mmol /L EDTA) was added and preserved in －20 ℃ refriger-
ator for later use．
Table 1 Buckwheat varieties used in this study
Serial
number
Name Species name Ploidy Symbol Provenance Origin Notes
1 Pinglu Common F． esculentum 2x ES1 Shanxi Shan Fang
Buckwheat
2 Chuntian 7 F． esculentum 2x ES2 Guiyang Chen Qing-fu
3 Zongtian 1 F． esculentum 2x ES3 Guiyang Chen Qing-fu
4 Pingqiao 2 F． esculentum 2x ES4 Gansu Wang Zong-sheng
5 Fengtian 1 F． esculentum 2x ES5 Guiyang Chen Qing-fu
6 Liutian 1 F． esculentum 2x ES6 Liupanshui Mao Chun
7 Sobano F． esculentum 2x ES7 Germany FJ Zeller Cross parent of
Fengtian 1，Zongtian 1
and Chuntian 7
8 Yanhe Common F． esculentum 2x ES8 Yanhe Chen Qing-fu Cross parent
Buckwheat of Fengtian 1
9 Gaoyuanbaihua F． esculentum 2x ES9 Liangshan of Weining Mao Chun Pedigree parent
Common Buckwheat of Weitian-2
10 Weitian-2 F． esculentum 2x ES10 Weining Mao Chun
11 Gaoyuanhonghua F． esculentum 2x ES11 Xueshan of Weining Mao Chun Pedigree parent
Common Buckwheat of Weitian-1
12 Weitian-1 F． esculentum 2x ES12 Weining Mao Chun
13 Jiujiang Tartary F． tataricum 2x TA1 Jiangxi Mao Chun
Buckwheat
14 Defangximiqiao F． tataricum 2x TA2 Panxian Zhang Qing-ming Pedigree parent
of Liuku 2081
15 Liuku 2081 F． tataricum 2x TA3 Shuicheng Zhang Qing-ming
16 Meihuashan Tartary F． tataricum 2x TA4 Meihuashan of Weining Mao Chun Pedigree parent
Buckwheat of Weiku-2
17 Weiku-2 F． tataricum 2x TA5 Weining Mao Chun
18 Mazha Tartary F． tataricum 2x TA6 Mazha of Weining Mao Chun Pedigree parent
Buckwheat of Weiku-1
19 Weiku-1 F． tataricum 2x TA7 Weining Mao Chun
RAPD amplification reaction The RAPD amplification re-
action system ( total volume 20．0 μl) contained 2 μl 10 × PCR
buffer，1．5 μl 25 mmol /L MgCl2，0．4 μl 10 mmol /L dNTP，1．0 μl
4 μmol /L 10-base RAPD primers，0．2 μl 2．5 U/μl Taq DNA pol-
ymerase，4．0 μl template DNA ( 25 ng/μl) and 10．9 μl ddH2O．
The PCR program was started with predenaturing at 94
℃ for 5 min，followed by 37 cycles． Each cycle included de-
naturing at 94 ℃ for 1 min，annealing at 37 ℃ for 1 min，and
extension at 72 ℃ for 2 min; the amplification was completed by
holding the reaction mixture at 72 ℃ for 5 min to allow complete
extension of PCR products． PCR amplification product was de-
tected with 0． 8% agarose gel electrophoresis ( containing 0． 5
μg/ml ethidium bromide) using λDNA/EcoRⅠ+HindⅢ as mo-
lecular weight standard; after electrophoresis，the gel was ob-
served and photographed under ultraviolet light．
Data Analysis
UPGMA method was employed for data analysis． For
each sample，the presence of RAPD amplification band was
recorded as 1，while the absence of RAPD amplification
band was recorded as 0 ． The NTSYS-pc 2． 10 e software
was used for statistical analysis．
According to the statistical result of all the amplification
bands，the bands stably and significantly different from others
were identified as characteristic bands of each variety，and
used to draw the fingerprint of each variety． Then，the bands
of all the common and tartary buckwheat varieties were com-
pared，to find out all bands specific to each species and sig-
nificantly different from the other species as the characteristic
bands of each species，to draw the fingerprints of common
and tartary buckwheat．
Results and Analysis
RAPD amplification
The seven primers and RAPD amplification result of the
19 materials were shown in Fig． 1 and Table 2． It could be
concluded from Table 2 that a total of 149 amplified DNA
bands were obtained． In which，bands amplified by different
primers differed from 9 to 29，and the average number was
21． 29． There were a total of 141 polymorphic bands; poly-
morphic bands of different primers ranged in 9 －29，and the
average number polymorphic bands was 20． 14． The ratio of
polymorphic bands was 88． 46% － 100%，with the average
of 94． 89%．
Table 2 The sequences of RAPD primers and their polymorphic band
numbers
Primer Primer sequence Numberof bands
Number of
polymorphic
bands
Rate of
polymorphic
bands∥%
s52 5 CACCGTATCC 21 19 90． 48
s42 5 GGACCCAACC 19 18 94． 74
s43 5 GTCGCCGTCA 28 27 96． 43
s69 5 CTCACCGTCC 26 23 88． 46
s353 5 CCACACTACC 17 16 94． 12
s428 5 ACCTCAGCTC 9 9 100． 00
s1182 5 ACCCTCGGAC 29 29 100． 00
Total 149 141
Average 21． 29 20． 14 94． 89
Cluster Analysis
It could be concluded from Fig． 2 that，as T =0． 60，the
19 buckwheat varieties could be divided into two groups，
namely，common buckwheat and tartary buckwheat; as T =
66 Agricultural Science ＆ Technology Vol． 12，No．1，2011
0．73，the 19 buckwheat varieties were divided into group 1
( tartary buckwheat ) ，group 2 ( ES2 － 12 ) and group 3
( ES1) ． The result suggested that in the tested buckwheat va-
rieties，the genetic variation of common buckwheat was grea-
ter than that of tartary buckwheat; in addition，ES1 had a dis-
tant genetic relationship with other buckwheat varieties． When
T =0． 97，the tested buckwheat varieties were divided into 12
groups． In which， the common buckwheat included nine
groups，namely，ES9 － ES12 was divided into a group，and
each of ES1 － ES8 separately formed a group; the result
showed a close genetic relationship among ES9 －ES12，while
a distant genetic relationship among ES1 － ES8． Tartary
buckwheat could be divided into three groups． The TA3 －TA7
was considered as a group，while each of TA1 and TA2 was
identified as a group． The results suggested that TA3 － TA7
had a closer genetic relationship to each other，while TA1 and
TA2 were a distant genetic relationship from the TA3 － TA7．
In addition，the intraspecific genetic difference among com-
mon buckwheat was greater，while that among tartary buck-
wheat was relatively smaller．
M: Marker; A －G: RAPD DNA bands by primers S52，S42，S43，S69，S353，S428 and S1182，respectively; 1 －19: RAPD bands of
ES1，ES2，ES3，ES4，ES5，ES6，ES7，ES8，ES9，ES10，ES11，ES12，TA1，TA 2，TA 3，TA 4，TA 5，TA6 and TA7，respectively．
Fig． 1 The pattern of RAPD DNA bands of the tested buckwheat materials
Fig． 2 Phylogenetic tree of 19 buckwheat varieties based on
RAPD
Fingerprint analysis
It could be concluded from the statistical result that each
of the varieties showed specific bands，suggesting that these
buckwheat varieties differed from each other to a certain ex-
tent． The bands of a certain common buckwheat variety differ-
ent from the bands of other varieties were considered as the
characteristic bands of the variety to construct RAPD finger-
print of common buckwheat varieties ( Fig． 3) ． The bands of
each tartary buckwheat variety different from the bands of oth-
er varieties was considered as the characteristic bands of the
variety to construct RAPD fingerprint of tartary buckwheat va-
rieties ( Fig． 4 ) ． The different bands between common and
tartary buckwheat varieties were compared to construct the
76DENG Lin-qiong et al． RAPD Analysis for Genetic Diversity of Nineteen Common and Tartary Buckwheat Varieties
RAPD fingerprint of common and tartary buckwheat varieties
( Fig． 5) ． It could be concluded from Fig． 3 －5 that the poly-
morphism within different varieties of common buckwheat was
higher than that of tartary buckwheat． The intraspecific differ-
ence and polymorphism of different buckwheat varieties were
relatively low，and thus there were more common bands of
buckwheat varieties．
Fig． 3 The RAPD fingerprint map of common buckwheat varieties
Fig． 4 The RAPD fingerprint map of tartary buckwheat varieties
Fig． 5 The species RAPD fingerprint map of common and tar-
tary buckwheat
Discussions
At present，there are some researches on buckwheat ge-
netic diversity all over the world． WANG Li-hua et al．［14］ ana-
lyzed the genetic diversity of Yunnan wild buckwheat re-
sources with RAPD method，and the result showed that the
interspecific genetic diversity of buckwheat species was more
abundant than their intraspecific genetic diversity． The geno-
type of 10 cultivated tartary varieties was studied by TAN Ping
et al．［20］，and it was found that the genetic similarity coeffi-
cient among the genotypes was 0． 184 6 －0． 852 5，indicating
a high degree of genetic similarity，so it was suggested that
the parent materials of selective breeding of buckwheat should
be expanded． FAN Dong-li et al．［21］ detected 30 local buck-
wheat varieties using RAPD，and found significant genetic
difference of buckwheat at the molecular level． RAPD analysis
on 52 local buckwheat resources and cultivars and 1 wild vari-
ety was performed by Sharma et al．［22］，and the results
showed that the similarity coefficients of the buckwheat varie-
ties ( not including wild variety) ranged from 0． 61 to 1． 00．
Kump et al．［23］using RAPD technique studied the genetic di-
versity of 40 buckwheat materials ( cultivated types and wild
types) from Tibetan Plateau and the surrounding areas，and
found the genetic polymorphism among populations was high-
er than that within population． In present study，the genetic di-
versity of 19 cultivars of common buckwheat and tartary buck-
wheat was analyzed using RAPD technique，149 DNA amplifi-
cation bands were obtained，in which，there were 141 poly-
morphic bands，and the average percentage of polymorphic
bands was 94．89%． Polymorphism analysis and cluster anal-
ysis showed that the buckwheat varieties were different from
each other． All cultivars showed some differences from each
other． The common buckwheat varieties native to Weining
shared a closer genetic relationship with each other，while
other buckwheat varieties were distantly related． A certain de-
gree of intraspecific genetic diversity respectively existed in
common and tartary buckwheat; the common buckwheat spe-
cies shared a higher intraspecific genetic diversity than tartary
buckwheat; however， the intraspecific genetic diversity of
both common and tartary buckwheat was lower than interspe-
cific genetic diversity，and the result was consistent with the
previous research． In addition，the present study also found
that Difangximiqiao ( TA2) as the parent of Liuku 2081 ( TA3)
shared a distant relationship with Liuku 2081 in the clustering
tree; however，Weiku-1 ( TA7) was closely related to Liuku
2081 ( TA3) ，the reason still needs to be further researched．
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resources and crop evolution，2002，49( 6) : 565 －572．
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WU Xiao-yan
甜荞和苦荞品种遗传多样性的 RAPD分析( 摘要)
邓琳琼1，2，张 奎1，黄凯丰1，陈庆富1* ( 1．贵州师范大学生命科学学院植物遗传育种研究所，贵州贵阳 550001; 2．毕节学院岩溶山
区生态建设研究所，贵州毕节 551700)
［目的］对甜荞和苦荞品种遗传达室的特性进行了 RAPD分析。
［方法］以 7个随机引物对贵州省 1999 ～2010年荞麦区甜荞和苦荞参试品种及其亲本等合计 19个品种进行了 RAPD分析。
［结果］共获得 149条 DNA扩增带，其中多态性谱带 141条，多态性谱带的平均比率为 94． 89%。多态性分析及聚类分析表明，供试品种彼此
均有一定的差异，其中威宁甜荞品种彼此新缘关系较近缘，而其他甜荞品种间关系较远。遗传变异性程度，以种间差异最大，其次是甜荞种
内不同品种间，而苦荞种内不同品种间的遗传变异性最小。
［结论］该研究初步建立了 19个品种的 RAPD指纹图谱。
关键词 甜荞;苦荞; RAPD; 遗传多样性
基金项目 麦荞麦国家十二·五现代农业产业体系荞麦育种岗位科学家资金;贵州省动植物育种专项［黔农育专字 ( 2010) 023 号］;国家自然科学基
金( 31060207) ;贵州省农业攻关项目［黔科合 NY字( 2010) 3094］。
作者简介 邓琳琼( 1982 － ) ，女，湖南武冈人，硕士研究生，研究方向: 植物遗传、起源、进化。* 通讯作者
收稿日期 2011-01-21 修回日期 2011-02-18
96DENG Lin-qiong et al． RAPD Analysis for Genetic Diversity of Nineteen Common and Tartary Buckwheat Varieties