全 文 ：Vol. 30 , No. 11
pp. 1097 - 1101 　Nov. , 2004
作 　物 　学 　报
ACTA AGRONOMICA SINICA
第 30 卷 第 11 期
2004 年 11 月 　1097～1101 页
Analysis of DNA Polymorphism and Genetic Relationships in Cultivated Peanut
( Arachis hypogaea L. ) Using Microsatellite Markers
HAN Zhu2Qiang1 , GAO Guo2Qing2 , 3 , WEI Peng2Xiao3 , TANG Rong2Hua1 , ZHONG Rui2Chun1
(1 Cash Crop Institute of Guangxi Academy of Agricultural Science , Nanning 530007 , Guangxi ; 2 Guangxi Crop Genetic Improvement and Biotechnology Laboratory ,
Nanning 530007 , Guangxi ; 3Biotechnology Research Center of Guangxi University , Nanning 530005 , Guangxi , China)
Abstract 　Eleven SSR primer pairs were used to amplify the genomic DNA isolated from 24 peanut genotypes including all
4 market types. Four primers could detected polymorphism and produced a total of 33 alleles. The average number of alle2
les per locus was 8. 25 , and up to 13 alleles were found at one locus. The genetic similarity ( GS) indexes among 24 culti2
vated peanut accessions were calculated based on the data from these SSR markers. The value of GS was varied from 0. 2
to 1. 0 , with an average of 0. 4788. The results of UPGMA indicated that the majority of 24 accessions could be divided into
two groups by subspecies , and most genotypes were clustered by market types. It is no doubt that microsatellites are very
useful DNA markers to analyze DNA polymorphism and genetic relationship in cultivated peanut .
Key words 　Microsatellites ; Peanut ; Polymorphism; Genetic relationship
利用 SSR标记分析栽培种花生多态性及亲缘关系
韩柱强1 　高国庆2 , 3 　韦鹏霄3 　唐荣华1 　钟瑞春1 Ξ
(1 广西农业科学院经济作物研究所 ,广西南宁 530007 ;2 广西作物遗传改良生物技术重点开放实验室 ,广西南宁 530007 ;3 广西大学生物技术实
验中心 ,广西南宁 530005)
摘 　要 　利用 11 对 SSR 引物对 24 个花生栽培品种 (包括四大类型)进行 PCR 扩增分析 ,其中 4 对检测到明显的多态性 ,
共检测到 33 个等位基因变异 ,每一个位点上检测到的等位变异数为 5～13 个 ,平均为 8. 25 个。根据扩增结果可以将 24
个品种中的 21 个相互区分。供试品种间的遗传相似系数值为 0. 2～1. 0 ,平均为 0. 4788。根据 UPGMA 聚类分析结果 ,供
试品种大多数按亚种聚为两大类群 ( Ⅰ、Ⅱ) ;在两大类群下 ,大多数品种也基本上按类型分类。本研究结果表明 ,SSR 在
分析栽培种花生 DNA 多态性和遗传关系方面非常有用。
关键词 　SSR ;花生 ;多态性 ;遗传关系
中图分类号 : S565
　　Cultivated peanut or groundnut ( Arachis hypogaea
L. ) is an important crop for oil and protein source. In
China , two major market types are widely grown , Virginia
in the North and Spanish in the South. The other two
types , Valencia and Runner are rarely scattered across
small area. Despite the existence of substantial diversity
among cultivated peanut genotypes for various morphologi2
cal , physiological and agronomic traits , very little DNA
variations have been detected by using protein2 or DNA2
based markers[1～5 ] . However , recent studies showed that
AFLP , RAPD and SSR markers could detect considerable
polymorphism in a limited number of accessions of culti2
vated peanut [6～14 ] , which hold a promise for marker2
based genetic improvement of peanut , as in most other
crops.
Microsatellites , also known as simple sequence re2
peats or SSRs , are small array of one to six tandemly ar2
ranged bases spread throughout the genomes. Microsatel2
lites as markers are advantageous over many other markers
mainly because they are highly polymorphic , highly abun2
dant , co2dominant inheritance , analytical simple and
readily transferable. SSRs have now been recommendedΞFoundation items : Supported by Guangxi Science Foundation (No. Guikejj 0236038 and No. Guikehui 0342003 ) .
Biography :Han Zhu2qiang(1968 - ) , master , major in peanut genetic breeding. 3 Corresponding auther : GAO Guo2Qing.
Received(收稿日期) : 2003207230 ,Accepted(接受日期) :2003212201.

as standard markers to be used in the construction of
highly saturated maps , and in some cases , in marker2as2
sisted selection. Level of polymorphism detected by mic2
rosatellites is generally high in almost all plants assayed.
In peanut , however , SSR markers were not available until
He et al . succeeded in developing SSR primers in large
scale through microsatellite2enriched library[15 ] . The ob2
jectives of this study were to detect DNA variation in Chi2
nese peanut germplasm and to study genetic relationship
among closely2related cultivated peanut genotypes using
their developed SSR markers.
1 　Materials and Methods
1. 1 　Plant materials
　　Twenty2four peanut genotypes representing four mar2
ket types from the bank of Guangxi peanut germplasm
were used for the analysis. Among them , five were breed2
ing lines , 11 were local germplasms and 8 were intro2
duced accessions (Table 1) . Genomic DNA was extracted
from leaves of the 152day2old seedlings by a modified
CTAB method.
Table 1 Peanut accessions used for study
No. Accession Origin Type No. Accession Origin Type
1 BC22420221 Guangxi , China Spanish 13 Lipu fanguidou(荔浦番鬼豆) Guangxi , China Virginia
2 Xie kang qing(协抗青) Hubei , China Spanish 14 Pingguo da huasheng(平果大花生) Guangxi , China Virginia
3 Hei huasheng(黑花生) Hubei , China Spanish 15 Baise da huasheng(百色大花生) Guangxi , China Virginia
4 Guang liu(广柳) Guangxi , China Spanish 16 Yangshuo fuli yangdou(阳朔福利洋豆) Guangxi , China Virginia
5 BC22454220 Guangxi , China Spanish 17 Parapoto India Virginia
6 5084 Guangxi , China Spanish 18 Miandian Burma Virginia
7 Xiao huasheng(小花生) Guangxi , China Runner 19 NCAC17090 India Valencia
8 Ningming wuqu zhihang(宁明五区峙行) Guangxi ,China Runner 20 PI341879 USA Valencia
9 Beiliu qindou(北流钦豆) Guangxi ,China Runner 21 Tifrust212 USA Valencia
10 Long’an baowan huasheng(隆安保湾花生) Guangxi , China Runner 22
Rwanda huasheng
(卢旺达花生) Rwanda Valencia
11 Mashan hequn huasheng(马山合群花生) Guangxi , China Runner 23 Tifrust26 USA Valencia
12 Shanglin tangjiang huasheng(上林塘江花生) Guangxi , China Runner 24 ICG1703 India Valencia
1. 2 　PCR and electrophoresis
SSR primers were developed and kindly provided by
Dr. Guohao He (Tuskegee University , USA) . PCR amp2
lification was performed in 10μL reaction mixture con2
taining 1 unit of Taq DNA2polymerase , 10 ×PCR buffer
containing 1. 5 mmolΠL MgCl2 , 100 μmolΠL of each
dNTPs , 1. 5 pmolΠL of forward and reverse primers and
50 ng of genomic DNA. Amplification was carried out on
a Biometra T1 Thermocycler using the following program :
an initial denaturation of 4 min at 94 ℃, 30 cycles of 1
min at 94 ℃, 1 min at 55 ℃, 2 min at 72 ℃, and final
extension at 72 ℃ for 10 min. The PCR products were
separated by 6 % denatured polyacrylamide gel electro2
phoresis and visualized by silver staining.
1. 3 　Data Analysis
Genetic similarity ( GS) was calculated according to
the formula : GS ij = 2 N ijΠ( N i + N j ) , where N ij was the
band number that appeared in both genotypes i and j . N i
and N j were the total band number of genotypes i and j ,
respectively. Genetic diversity ( GD) was calculated as :
GDij = 1 - GS ij . Clustering analysis was carried out ac2
cording to unweighted pair group method arithmetic ave2
rages (UPGMA) by using SAS System Software.
2 　Results and Discussion
2. 1 　Polymorphism among cultivated peanut detected
by SSR markers
　　Eleven SSR primers were screened and four of them
were able to detect 33 alleles among 24 peanut genotypes.
For the four polymorphic loci , a range of 5 - 13 alleles
were obtained , with an average of 8. 25 per locus (Table
2) . One primer pair , PM236 , detected as many as 13 al2
8901 　　　作 　　物 　　学 　　报 30 卷 　

leles among the cultivated peanut with diversified origin
(Fig. 1) . Even within the same market type , DNA fin2
gerprints were different ( Fig. 2) . By employing the four
polymorphic markers , 21 out of the 24 peanut genotypes
could be clearly differentiated. The results indicated that
the number of alleles detected by peanut SSR primers
could be comparable to many other plants , such as soy2
bean , rice , barley and cucumber , and microsatellites
could be one of the most powerful tools for differentiating
closely2related peanut cultivars. However , more than half
of the primers failed to detect any polymorphism , while
these primers could identify genetic variation among the
peanut accessions collected from worldwide[15 ] , which in2
dicated the genetic base of Chinese peanut germplasm was
narrow. More informative SSR markers are apparently
needed to be developed for the construction of highly satu2
rated maps and marker2assisted selection in cultivated
peanut .
Table 2 Number of alleles detected by four SSR primers
Primer Annealing temperature Number of alleles detected
PM250 55 ℃ 5
PM215 55 ℃ 7
PM236 55 ℃ 13
PM231 52 ℃ 8
Fig. 1 Profile of amplification products from genomic DNAs of 24 genotypes using SSR primer PM236
Fig. 2 Profile of amplification products from genomic DNAs of 24 genotypes using SSR primer PM250
2. 2 　Genetic similarity and cluster analysis
GS values among 24 genotypes based on SSR
analysis ranged from 0. 2 to 1. 0 (Table 3) , with an aver2
age of 0. 48. Genotype 13 , 15 and 16 had the highest
values of GS ( GS = 1) between each other , and all of
them were Virginia. The lowest genetic similarity showed
between genotype 23 vs. 7 , 9 , 13 , 14 , 15 and 16 re2
spectively with GS value of 0. 2 , where genotype 23 is of
ssp . f astigiata Waldron but the other accessions are ssp .
hypogaea .
9901　11 期 HAN Zhu2Qiang et al . :Analysis of DNA Polymorphism and Genetic Relationships in Cultivated Peanut111111 　　　

Table 3 Genetic similarity among 24 accessions based on SSR analysis
Material 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 1
2 0. 381 1
3 0. 700 0. 526 1
4 0. 818 0. 476 0. 700 1
5 0. 800 0. 421 0. 556 0. 800 1
6 0. 667 0. 600 0. 632 0. 667 0. 842 1
7 0. 400 0. 211 0. 444 0. 400 0. 444 0. 316 1
8 0. 696 0. 364 0. 667 0. 696 0. 571 0. 455 0. 667 1
9 0. 400 0. 211 0. 333 0. 400 0. 444 0. 316 0. 889 0. 381 1
10 0. 500 0. 316 0. 444 0. 500 0. 556 0. 421 0. 778 0. 286 0. 889 1
11 0. 696 0. 455 0. 667 0. 696 0. 571 0. 455 0. 476 0. 583 0. 571 0. 667 1
12 0. 400 0. 211 0. 333 0. 300 0. 444 0. 421 0. 667 0. 286 0. 667 0. 556 0. 381 1
13 0. 300 0. 316 0. 333 0. 300 0. 333 0. 316 0. 667 0. 286 0. 778 0. 667 0. 571 0. 667 1
14 0. 300 0. 316 0. 333 0. 300 0. 333 0. 316 0. 667 0. 286 0. 667 0. 556 0. 476 0. 667 0. 889 1
15 0. 300 0. 316 0. 333 0. 300 0. 333 0. 316 0. 667 0. 571 0. 778 0. 667 0. 571 0. 667 1. 000 0. 889 1
16 0. 300 0. 316 0. 333 0. 300 0. 333 0. 316 0. 667 0. 571 0. 778 0. 667 0. 571 0. 667 1. 000 0. 889 1. 000 1
17 0. 696 0. 273 0. 571 0. 783 0. 667 0. 545 0. 286 0. 667 0. 381 0. 476 0. 667 0. 190 0. 286 0. 190 0. 286 0. 286 1
18 0. 571 0. 300 0. 421 0. 667 0. 737 0. 600 0. 316 0. 273 0. 421 0. 526 0. 545 0. 211 0. 316 0. 211 0. 316 0. 316 0. 909 1
19 0. 526 0. 444 0. 471 0. 632 0. 588 0. 444 0. 353 0. 400 0. 353 0. 471 0. 500 0. 235 0. 235 0. 235 0. 235 0. 235 0. 700 0. 778 1
20 0. 800 0. 316 0. 667 0. 700 0. 667 0. 526 0. 333 0. 667 0. 333 0. 444 0. 667 0. 333 0. 222 0. 222 0. 222 0. 222 0. 762 0. 632 0. 706 1
21 0. 556 0. 353 0. 500 0. 667 0. 750 0. 706 0. 375 0. 632 0. 500 0. 625 0. 526 0. 375 0. 500 0. 375 0. 500 0. 500 0. 632 0. 706 0. 400 0. 375 1
22 0. 353 0. 375 0. 400 0. 471 0. 400 0. 375 0. 267 0. 333 0. 267 0. 400 0. 333 0. 267 0. 267 0. 267 0. 267 0. 267 0. 333 0. 375 0. 571 0. 400 0. 462 1
23 0. 636 0. 381 0. 700 0. 545 0. 500 0. 571 0. 200 0. 174 0. 200 0. 300 0. 522 0. 300 0. 200 0. 200 0. 200 0. 200 0. 435 0. 286 0. 316 0. 600 0. 333 0. 471 1
24 0. 700 0. 421 0. 778 0. 600 0. 556 0. 632 0. 222 0. 571 0. 222 0. 333 0. 571 0. 333 0. 222 0. 222 0. 222 0. 222 0. 571 0. 421 0. 471 0. 778 0. 375 0. 800 0. 800 1
　　Dendrogram constructed from SSR data showed that
24 peanut accessions were divided into two clusters Ⅰ
and Ⅱ distinctly , corresponding to the two subspecies
hypogaea and f astigiata , with exception of 4 genotypes
(Fig. 3) . Cluster Ⅰcontained 8 peanut accessions which
belong to ssp . hypogaea , and most of them were formed
into two subgroups ( Ⅰ21 and Ⅰ22 ) by botanical
varieties. The average genetic diversity ( GD) was similar
in subgroup Ⅰ21 and subgroup Ⅰ22 , with the value of
0. 167 and 0. 148 , respectively. Cluster Ⅱconsisted of
16 accessions , 12 of them from ssp . f astigiata and the
others from ssp . hypogaea . The accessions in this cluster
could also be divided into two subgroups ( Ⅱ21 and Ⅱ2
2) , which primarily complied with their botanical types.
Five of all 6 Spanish accessions were in subgroup Ⅱ21
and half of the Valencia accessions were in Ⅱ22. Com2
paring with the cluster Ⅰ, the average genetic diversity
( GD) value in cluster Ⅱwas higher , with an average
value of 0. 4405. This was probably because that the five
breeding lines from hybridization , 8 introduced accessions
from foreign countries included in this cluster might be far
related.
Fig. 3 Dendrogram showing the genetic relations among 24 cultivated peanut genotypes
　　The results indicated that SSRs were very effective
DNA markers on genetic similarity and cluster analysis in
cultivated peanut . By using 4 primer pairs , 24 accessions
could be divided into two clusters and four groups corre2
0011 　　　作 　　物 　　学 　　报 30 卷 　

sponding to two subspecies and four market types. In con2
clusion , the genetic relationships between accessions can
be revealed by SSR markers. More SSR markers should
be developed , not only for diversity studies , but also for
high2resolution genetic mapping , evolutionary studies and
marker2assisted selection in peanut .
Acknowledgements : The authors thank Guangxi Crop Ge2
netic Improvement and Biotechnology Lab for providing re2
search facilities. Special thanks are given to Dr. Yang
Yong2zhong for assistance with the data analysis.
References
[1 ] Kochert G A , Halward T M , Branch W D , Simpson C E. RFLP varia2
bility in peanut ( Archis hypogaea L. ) cultivars and wild species. Theor
Appl Genet , 1991 , 81 :565 - 570
[2 ] Halward T M , Stalker H T , Larue E A , Kochert GA. Genetic variation
detectable with molecular markers among unadapted germplasm resources
of cultivated peanut and related wild species. Genome , 1991 , 34 :
1 013 - 1 020
[3 ] Halward T M , Stalker H T , Larue E A , Kochert G A. Use of single2
primer DNA amplification in genetic studies of peanut ( Arachis hypogaea
L. ) . Plant Mol Biol , 1992 , 18 : 315 - 325
[4 ] Paik2Ro O G, Smith R L , Kochert D A. Restriction fragment length
polymorphism evaluation of six peanut species within the Arachis section.
Theor Appl Genet , 1992 , 84 :201 - 208
[5 ] Stalker H T , Philips T G, Murphy J P , Jones T M. Diversity of isozyme
patterms in Archis species. Theor Appl Genet , 1994 , 87 :746 - 755
[ 6 ] He G H , Prakash C S. Identification of polymorphic DNA markers in cul2
tivated peanut ( Archis hypogaea L. ) . Euphytica , 1997 , 97 :143 - 149
[ 7 ] Weng Y2J (翁跃进) , Santosh Gurtu , Nigan S N. Fingerprinting in
groundnut using AFLP. Chinese Journal of Oil Crop Sciences (中国油料
作物学报) ,1999 ,21 (1) :10 - 12
[8 ] Yie B2Y(叶冰莹) ,Chen Y2Q(陈由强) ,Zhu J2M(朱锦懋) . Analysis
of genetic relationship among peanut cultivars ( Arachis hypogaea) . Chi2
nese Journal of Oil Crop Sciences (中国油料作物学报) ,1999 ,21 (3) :
15 - 18
[9 ] Subramanian V , Gurtu S , Nageswara Rao R C , Nigam S N. Identifica2
tion of DNA polymorphism in cultivated groundnut using random amplified
polymorphic DNA (RAPD) assay. Genome , 2000 ,43 (4) :656 - 660
[ 10 ] Jiang H2F(姜慧芳) ,Ren X2P(任小平) . RAPD in cultivated groundn2
ut ( Arachis hypogaea L. ) . Journal of Peanut Science (花生学报) ,
2002 ,31 (2) :10 - 13
[11 ] Hopkins M S , Casa A M , Wang T , Mitchell S E , Dean R E , Kochert
G D , Kresovich S. Discovery and characterization of polymorphic simple
sequence repeat (SSRs) in peanut. Crop Science ,1999 , 394 : 1 243 -
1 247
[12 ] Subramanian V , Gurtu S , Nageswara Rao R C , Nigam S N. Identifica2
tion of DNA polymorphism in cultivated groundnut using random ampli2
fied polymorphic DNA (RAPD) assay. Genome , 2000 , 43 (4) :656 -
660
[13 ] Raina S N , Rani V , Kojima T , Ogihara Y, Singh K P , Devarumath R
M. RAPD and ISSR fingerprints as useful genetic markers for analysis of
genetic diversity , varietal identification , and phylogenetic relationships
in peanut ( Arachis hypogaea L. ) cultivars and wild species. Genome ,
2001 , 44 (5) :763 - 772
[ 14 ] He G H , Prakash C S. Evaluation of genetic relationships among botani2
cal varieties of cultivated peanut ( Arachis hypogaea L. ) using AFLP
markers. Genetic Resources and Corp Evolution , 2001 , 48 :347 - 352
[15 ] Guohao He , Ronghua Meng , Melanie Newman , Guoqing Gao , Roy N
Pittman , Prakash C S. Microsatellites as DNA makers in cultivated pea2
nut ( Arachis hypogaea L. ) . BMC Plant Biology , 2003. 3
欢迎订阅 2005 年《玉米科学》
《玉米科学》是 1992 年经国家新闻出版署和国家科技部批准出版的全国性科技期刊。近年来 ,玉米科学
已经发展成为我国惟一的玉米学术刊物 ,在国内外玉米界具有较大影响。2004 年被评为中文核心期刊。
《玉米科学》是理论与实践相结合、普及与提高相结合的刊物。主要报道 :遗传育种、品种资源、耕作栽
培、生理生化、生物工程、土壤肥料、专家论坛、国内外玉米科研动态、新品种信息等方面的内容。适合科研、
教学、生产及管理方面的人员参考。
《玉米科学》为季刊 ,季末月 25 日出版。国际大开本 ,120 页Π期 ,定价 10 元 ,全年 40 元。国内外公开发
行 ,邮发代号 :122137 ,全国各地邮局 (所) 均可订阅 ,漏定者可直接向编辑部补定。地址 :吉林省公主岭市西
兴华街 6 号 ,邮编 :136100。
《玉米科学》刊登广告 ,广告经营许可证 :西广字 050104 号。有意者请电话联系 :043426283137 , E2mail :
ymkx @cjaas. com
1011　11 期 HAN Zhu2Qiang et al . :Analysis of DNA Polymorphism and Genetic Relationships in Cultivated Peanut111111