全 文 ：Analysis of Genetic Structure of Natural Populations of
Castanopsis fargesii by RAPDs
ZHUQi_Hui , PAN Hui_Xin , ZHUGE Qiang , YIN Tong_Ming , ZOU Hui_Yu , HUANG Min_Ren＊
(Key Laboratory of Forest Tree Genetics and Gene Engineering , Nanjing Forestry University , Nanjing 210037, China)
Abstract:　Genetic diversity and population genetic structure in 188 individuals from five natural populations
of Castanopsis fargesii Franch.were studied by RAPD markers.Three hundred and eighty_five loci were iden-
tified with 41 oligonucleotide primers , out of which 157 loci were polymorphic and accounted for 40.78% of
total genetic diversity at species level.Shannon s indices of diversity (I)and Nei s gene diversity(h)were
0.459 7 and 0.296 at the species level , respectively .The result showed that genetic variation of C.fargesii
populations mainly existed within populations.Genetic differentiation (Hsp_Hpop)/Hsp estimated with Shan-
non s index of diversity and coefficient of gene differentiation(Gst)were 0.047 6 and 0.042 9 respectively ,
which were confirmed by the analysis of molecular variance(AMOVA).Therefore , it is apparent that within_
population variation accounted for 94.97% and among_populations variation accounted for only 5.03% of the
total genetic diversity.AMOVA also indicated that there was significant differentiation among populations as
well as among individuals within a population.
Key words:　Castanopsis fargesii;natural populations;RAPD;genetic structure
　　Evergreen broad_leaved subtropical forests in China ,
with ample species and wide distribution , play an impor-
tant role in the forestry ecosystem all over the world.
Species in the genus of Castanopsis (Fagaceae)are main
components of evergreen broad_leaved forests in the sub-
tropical regions in central China , most of which have the
character of good quality of wood and are elite materials
for furniture and decoration.In addition , they have an
important role in the improvement of ecosystem and pro-
tection of biodiversity in the subtropical area.However ,
the evergreen broad_leaved forests have been seriously de-
stroyed in the subtropical of China and a lot of species
have disappeared or been on the brink of extinction be-
cause of the deforestation that is one of main reasons for
the damage to evergreen broad_leaved forests.On the oth-
er hand , since large areas of evergreen broad_leaved
forests have been replaced by needle_leaved forests , the
biodiversity has been threaten as a result of homogeneity
of the forests , which destroyed the sustainable develop-
ment of forestry.
Castanopsis fargesii is an important germplasm.As a
founder species in the east of the subtropics , its popula-
tion genetics has not been investigated thoroughly.RAPD
technique has been widely used in the study of forest ge-
netic diversity and genetic structure , such as Pinus
sylvestris (Szmidt et al , 1996), Populus tremuloides(Yeh et al , 1995), Picea abies (Bucci and Menozzi ,
1995), Eucalyptus globulus (Nesbitt et al , 1995),
Quertus petraea (Corre et al , 1997), Castanea dentate(Huang et al , 1998).In this study , the genetic diversity
of the well_reserved natural populations of C.fargesii in
the eastern China was evaluated.Our specific aims were:
(1)to detect the genetic variation within and between the
natural populations;(2)based on the above analysis , to
discuss which strategy should be selected for conserving
these germplasm resources of C.fargesii.
1　Materials and Methods
1.1　Plant materials
Castanopsis fargesii Franch.was sampled from five
populations in both Fujian and Jiangxi.Seeds from at
least 30 individuals in each population were collected in
October , 1997.In order to avoid the seeds from individu-
als in the same family , seed bearers were spaced at least
more than 50 m from each other.Seeds were stratified
and seedling was obtained in the following spring.Details
on collecting sites are available in Table 1.Fresh leaves
from 30-50 individuals in each population were harvest-
ed , and fast dried with silicon gels.
1.2　Extraction of total DNA
DNA was isolated following the method of Boom
et al (1990)with some modifications for C.fargesii.
One to two g of the dried leaves was submerged in liquid
nitrogen and then ground into powder , followed by addi-
tion of 2 mL of CTAB isolation solution , completely mix-
ing and incubation in water bath at 65 ℃ for 30-45min
and cooled immediately in ice water.Then 2 mL of chlo-
roform_isoamylol(24∶1)was added.After that , it was
mixed evenly and centrifuged at 10 000 r/min for 10min.
Finally 600μL of the supernatant was transferred to a 1.5
mL centrifuge tube with 75 μL of 12% silica_containing
water solution and vortexed for 5 s.It was kept at room
temperature for 10-15 min and centrifuged at 6 000
r/min for 15 s.The supernatant was discarded and the
Received:2002-02-27　Accepted:2002-08-29
Supported by the National Natural Science Foundation of China(39770628).
＊Author for correspondence.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(11):1321-1326
Table 1　The locality of Castanopsis fargesii populations sampled
No. Geographic location　　 Population　 Natural status　　　 Latitude/Longitude Individuals sampled
FJSW Shaowu , Fujian Redlight Tree Farm Pure plantation 27°21′/ 119°29′ 38
FJJO Jianou , Fujian Wanmulin Reserve Natural forest 27°03′/ 118°17′ 35
FJYK Yangkou , Fujian Yangkou Tree Farm Natural forest 26°52′/ 117°53′ 38
FJSX Saxian , Fujian Luoboyan Reserve Natural forest 26°26′/ 117°44′ 33
JXYY Yiyang , Jiangxi Yiyang Reserve Natural forest 28°26′/ 117°27′ 44
precipitate was washed with 600 μL of washing solution(12 g of GuSCN (pH 6.4)dissolved in 10 mL of 0.1
mol/L Tris_HCl buffer).Then it was centrifuged at 6 000
r/min for 15 s , which was duplicated once and followed
by washing twice with 600μL of 75% ethanol each.The
extract was dried naturally or by evaporation.One hun-
dred and fifty μL of 1×TE buffer was added , and then
vortexed for 5 s.After that it was kept in water bath at 56℃for 10 min , and centrifuged at 12 000 r/min for 10
min.The supernatant thus obtained was DNA solution
used in the experiment.The concentration of DNA was
determined by comparison with standard λDNA on an
agarose gel.
1.3　PCR amplification
A total of 1 024 arbitrary primers from Operon Tech-
nologies(Operon A_Z and AA_AZ kits)were used in the
RAPD analysis.DNA amplification was performed on a
Perkin_Elmer gene amplifier (PE9600).The conditions
and system for amplification followed the method de-
scribed by Yin et al(1999).The conditions included de-
naturation at 94 ℃ for 30 s , 38 cycles composed of de-
naturation at 94 ℃for 30 s , annealing at 40 ℃ for 30 s
and extension at 72 ℃for 1.5min , with a final extension
at 72 ℃ for 7 min and the PCR products were kept at
4 ℃.The reaction component (20 μL)contained 20
mmol/L Tris_HCl buffer(pH 8.3), 50μmol/L KCl , 500μg/mL BSA , 5 μL of 0.01% Geleton , 20 mmol/L
MgCl2 , 200μmol/L dNTP , approximately 10 ng of dou-
ble stranded DNA template and 1.0 U of Taq polymerase(produced by China Agricultural University).Amplifica-
tion products were analyzed by electrophoresis on 1.2%
agarose gels stained with ethidium bromide and pho-
tographed under ultraviolet light with a Polaroid photo-
graphic system.Molecular weights were estimated using a
100 bp DNA ladder.
1.4　Data analysis
The fragment datawere entered in a computer file as
binary matrix where 0 and 1 coded for absence and pres-
ence of a band , respectively.The phenotypic matrix was
assembled for further statistical analyses.In order to eval-
uate genetic variation with RAPDs , the percentage of
polymorphic bands (PPB) was calculated for each
primer.Also , average number of alleles(A), number of
effective alleles (Ne), Nei s gene diversity (h)and
Shannon s index of diversity (I)(Lewontin , 1972)were
calculated with the software POPGENE 32 (Yeh et al ,
1997).For the analysis of genetic structure , the program
AMOVA_PREP involved in the software package TFPGA
(Mark , 1997)was first used to calculate Euclidean dis-
tance(Excoffier et al , 1992)between individuals , and
the distance matrix was formed.Genetic variation was an-
alyzed by non_parameter Analysis of Molecular Variance(AMOVA)(Excoffier , 1993).Program version 1.5(Excoffier et al , 1992).In addition , the coefficient for
gene divergence (Gst)and gene flow (Nm)were calcu-
lated by POPGENE on the basis of gene frequencies.In
order to analyze genetic relationship among populations ,
UPGMA (unweighted pair group method arithmetic aver-
age)clustering was conducted according to Nei s genetic
distance (Nei and Li , 1979).At the same time , Mantel
test was applied to estimate the correlation between genet-
ic distance and geographic distance among populations.
2　Results
2.1　Primer screening
Because RAPD amplification is sensitive to reaction
condition , 1 024 primers were initially screened against
one plant randomly sampled from populations , and 351
primers which generated strong amplification products
were selected , and finally 41 primers (Table 2)which
produced clear and reproducible fragments were selected
for the further study.
2.2　Genetic diversity
2.2.1　Variation in phenotypic frequency　A total of
385 bands with molecular weight from 200 to 2 200 bp
were scored by 41 decamer primers , corresponding to an
average of 9.17 bands per primer.Of them , 157 bands
were polymorphic and the number of polymorphic bands
per primer ranged from 1 to 8 with an average of 3.74.
Figure 1 shows an example of polymorphic bands ampli-
fied by primer OPQ04.
Genetic diversity of every population was evaluated
with Shannon s index of diversity (Table 3).The diver-
sity for each primer ranged from 0.230 2 to 0.634 3 , and
was 0.449 7 , 0.438 2 , 0.444 9 , 0.432 8 and 0.425 9
respectively for five populations.
2.2.2 　Variation in gene frequency 　Each RAPD
band was treated as a locus with two alleles M , m respec-
tively , “1” represents genotype MM or Mm , “0” repre-
sents genotype mm.It is assumed that the gene frequency
within a population was under Hardy_Weinberg equilibri-
um.On this basis a gene frequency matrix was then cal-
culated.Bands with q2 <3/N (q was the frequency for
recessive genes and N was the sample size)were discard-
ed according to Lynch_Milligan (Lynch and Milligan ,
1999)method for calibrating RAPD data from a small
1322　 植物学报　Acta Botanica Sinica　Vol.44　No.11　2002
Table 2　Sequence of RAPD primers
Primer Sequence(5′-3′) Primer Sequence(5′-3′) Primer Sequence(5′-3′)
A04 AATCGGGCTG K07 AGCGAGCAAG AB07 GTAAACCGCC
A10 GTGATCGCAG K19 CACAGGCGGA AB16 CCCGGATGGT
A20 GTTGCGATCC L01 GGCATGACCT AE20 TTGACCCCAG
B01 GTTTCGCTCC L04 GACTGCACAC AF05 CCCGATCAGA
B04 GGACTGGAGT M05 GGGAACGTGT AH08 TTCCCGTGCC
B08 GTCCACACGG O20 ACACACGCTG AH13 TGAGTCCGCA
B12 CCTTGACGCA P01 GTAGCACTCC AN15 TGATGCCGCT
G02 GGCACTGAGG P13 GGAGTGCCTC AN16 GTGTCGAGTC
G11 TGCCCGTCGT Q04 GGTCACCTCA AP05 GACTTCAGGG
I07 CAGCGACAAG R19 CCTCCTCATC AP07 ACCACCCGCT
J01 CCCGGCATAA U03 CTATGCCGAC AQ02 ACCCGCGGTG
J12 GTCCCGTGGT U06 ACCTTTGCGG AQ03 GAGGTGTCTG
J15 TGTAGCAGGG W19 CAAAGCGCTC AU04 GGCTTCTGTC
J18 TGGTCGCAGA X01 CTGGGCACGA
Table 3　Genetic diversity of Castanopsis fargesii populations
Population Number of samples
Average number of
alleles(A)
Number of effective
alleles(Ne) Nei s gene diversity (h)
Shannon s index of
diversity(I)
FJSW 38 1.993 6 1.473 2 0.290 6 0.449 7
FJJO 35 1.987 3 1.460 6 0.282 4 0.438 2
FJYK 38 1.987 3 1.471 6 0.288 0 0.444 9
FJSX 33 1.961 8 1.456 6 0.279 5 0.432 8
JXYY 44 1.936 3 1.451 7 0.276 0 0.425 9
Population average
(SD)
37.6 1.973 3
(0.148 4)
1.462 7
(0.320 4)
0.283 3
(0.153 9)
0.438 3
(0.198 2)
Species 188 2.000 1.477 4 0.296 0 0.459 7
sample size.Genetic diversity was calculated by POP-
GENE 32.
It can be seen from Table 3 that Nei s gene diversity(h)at species level was 0.296 and Shannon s index of
diversity (I)was 0.459 7.While at population level ,
average number of alleles (A), number of effective alle-
les(Ne)of five populations were similar and the popula-
tion diversity in FJSW was the highest among five popula-
tions and the lowest in JXYY with h and I showing the
same tendency.
2.3　Genetic differentiation among populations
The AMOVA program was used to partition the ge-
netic variation by hierarchical analysis from the Euclidean
distance matrix.The result showed that the within_
population component accounted for 94.97% of the total
variation(Table 4).The among_population component ,
although accounting for only 5.03% of the variation , was
significantly different from zero (P <0.001).Similarly ,
coefficient of gene differentiation (Gst) calculated by
POPGEGE was only 0.042 9 , which indicated that the
great majority of genetic variation (95.71%) resided
within populations , and only a small amount of variation(4.29%) presented differences among populations.
There might be a high gene flow in C.fargesii , and Nm
was 11.26(Table 5).
Differentiation between populations evaluated with
Shannon s index of diversity (Hsp_Hpop)/Hsp was
0.047 6 , which was slightly higher than that estimated
with Gst(0.042 9), but a little lower than the result ob-
tained from AMOVA (0.050 3).Besides , the genetic
variation estimated from Shannon s index of diversity var-
ied among the primers , with OPL01 detecting the lowest(0.004 7)and OPG02 the highest(0.201 5).In addi-
tion to the primers of OPG02 , OPAE20 and OPAE07 ,
which had a value over 0.12 , all other 37 primers had a
value between 0.01 and 0.07(data not shown).
2.4　Genetic distance among populations
In order to reveal the relationships among popula-
tions , the Nei s genetic distance was calculated with
which a cluster analysis of this distance matrix based on
an UPAMA algorithm was used to generate a dendrogram(Fig.2).The dendrogram also indicated that genetic dis-
tance between populations was low (<0.03), and the
lowest was between FJJO and FJYK(0.004 3), the high-
est between FJSW and JXYY(0.027 9).Geographically
speaking , four populations from Fujian Province are locat-
ed in the northern part of Fujian Province and relatively
close to each other.But they are relatively far from
Yiyang of Jiangxi Province in geographical distance.It
could be seen from the dendrogram that population JXYY
was apparently in the outermost.Mantel test showed that
there was no significant correlation between genetic dis-
tance and geographic distance (r =0.778 8 , P =
0.103).
ZHU Qi_Hui et al:Analysis of Genetic Structure of Natural Populations of Castanopsis fargesii by RAPDs 1323　
Fig.1.　RAPD amplification products generated from primer OPQ04.
Table 4　Analysis of molecular variance of Castanopsis fargesii populations
　Source of variance d.f SSD MSD Variance component Percentage total(%) P_value ＊
Variance among regions 1 83.610 83.61 0.959 4 4.56 <0.001
Variance within regions 3 3 713.69 20.074 20.074 95.44 <0.001
Variance among populations 4 233.26 58.316 1.038 5.03 <0.001
Variance within populations 182 3 564.04 19.583 19.583 94.97 <0.001
＊ Signifi cance tests after 3 000 permutations.
d.f , degree of f reedom;MSD , expected mean squares;P_value , probabi lity of null distribution;SSD , sum of squares.
3　Discussion
Genetic diversity of a species is referred to the sum
of genetic information within the gene pool.Genetic data
are essential prerequisites for assessing the genetic struc-
ture of forest trees (Qian and Ge , 2001).Therefore ,
molecular techniques at DNA level have been widely used
in current studies.However , RAPD is a dominant marker
1324　 植物学报　Acta Botanica Sinica　Vol.44　No.11　2002
Table 5　Genetic differentiation in Castanopsis fargesii populations
　　　POPGENE
Ht Hs Gst Nm
Shannon index
Hsp Hpop (Hsp_Hpop)/Hsp
Species 0.295 9 0.283 3 0.042 9 11.263 5 0.469 6 0.448 3 0.047 6
SD 0.018 8 0.017 3 0.090 4 0.093 0
Gst , coefficient of gene differentiation;Hs and Hpop , gene diversity within populations;Ht and Hsp , gene diversity of species;Nm , gene flow.
Fig.2.　Dendrogram of five populations of C.fargesii generated by
UPGMA.
and has such disadvantages as incapability of distinguish-
ing marker/marker homozygotes from marker/null het-
erozygotes , difficulty in detecting multiple alleles and loss
of much information in testing genetic variation.It is sug-
gested that 2-10 times more individuals should be sam-
pled per RAPD locus than per RFLP or isozyme loci(Lynch and Milligan , 1994).In this study , we sampled
more than 30 individuals from every population in order to
reduce deviation.Krutovskii et al (1999)developed a
dominance simulation with which codominant population
data can be transformed to compare population genetic
statistics such as Hs , Ht and Gst more directly.They
pointed out that , after the simulating study from both
isozymes and RAPDmarkers in Douglas_fir which is wide-
ly distributed in North America , RAPD markers can
strongly underestimate population diversity but still rea-
sonably estimate population differentiation(Gst), if sam-
ple sizes are larger than 30 individuals.Therefore , sam-
ple sizes for every C.fargesii population were consistent
with the above statement.
Qian et al (2001)found that for dominant data
there was a significant correlation between φst_based
molecular variance analysis(AMOVA)and Nei s genetic
distance based on Hardy_Weinberg equilibrium , both of
which were suitable for analysis of genetic structure.
However , Nei s genetic distance should be calibrated by
Lynch_Milligan to reduce the influence of dominant mark-
ers which usually underestimated the genetic variation.It
is not necessary to make a Hardy_Weinberg assumption forφst_based molecular variance analysis , and the data for
genetic diversity could be analyzed by the significance
tests.Therefore , φst_based molecular variance analysis
was more reliable and could be chosen preferentially.
Analysis of Shannon s index of diversity had a similar re-
sult , which was closer to AMOVA and it could be used as
a supplement.The result in this paper also supported the
conclusion and the differentiations between populations
estimated by the methods mentioned above were close to
each other (Gst =0.042 9 , variation component =
0.050 3 , (Hsp_Hpop)/Hsp =0.047 6).In addition ,
the order of genetic diversity based onNei s gene diversi-
ty (h)and Shannon s index of diversity (I)is unani-
mous:FJSW>FJYK>FJJO>FJSX>JXYY(Table 3).
Although C.fargesii is a typical outcrossing plant , in
which self_cross is seldom found , it is appropriate to use
phenotypic frequency_based AMOVA program and Shan-
non index to measure genetic diversity.
Assessment of genetic diversity and population genet-
ic structure is essential in germplasm characterization and
conservation. By using 41 RAPD primers ,
157(40.78%)out of 385 assayed were polymorphic.
The genetic differentiation between populations based on
AMOVA(Table 4)was highly significant(P<0.001).
This level of among_population differentiation was also ob-
served at SSR loci in C.fargesii by Xu et al (2001)(Fst=0.031).There are many factors that have an ef-
fect on genetic structure among populations , of which se-
lection , mating system and gene flow are of the most im-
portance(Ge , 1998).Bussell(1999)made a summary
on RAPD studies of 35 species , and found that among 29
distant crossing species , the variation among populations
accounted for 19.3% of the total genetic variation(Gst=
0.193).The average Gst for six inbred species was
0.625.Hogbin and Peakall(1999)obtained the same re-
sult and found that the genetic variation of outcrossing
species mostly occurred within a population and the varia-
tion among populations usually accounted for less than
27%.Yeh et al (1995)made use of RAPD markers to
study the genetic variation of 249 individuals from eight
natural populations of Populus tremuloides and found that
there was a relatively high genetic variation in these popu-
lations as well as a significant genetic differentiation be-
tween populations (Gst =0.026).C.fargesii is out-
crossing and mostly perennial woody plant , and widely
distributed in the central subtropics including the area to
the south of Yangtze River.Therefore , such properties as
outcrossing trees with wide and continuous range , long
cycles of generations and strong ability of propagation re-
tain a considerable amount of genetic diversity and exhibit
little genetic differentiation among populations.
In terms of gene flow , it occurs frequently between
populations in C.fargesii (Nm=11.263 5).A modest
amount of gene flow would reduce the subdivision among
natural populations (Wright , 1978).Gene flow in C.
fargesii would occur mainly by means of migration and the
movement of such genetic carriers as pollens and seeds ,
which are two main forms for the spread (Hamrick ,
1987).C.fargesii is a species pollinated by insects.
Its nuts are sweet in taste and preferred by rodents.Its
seeds are spread mainly by gravity and animals trans-
portation.In addition , C.fargesii prefers moist soil and
the populations in this study are all near to water systems
ZHU Qi_Hui et al:Analysis of Genetic Structure of Natural Populations of Castanopsis fargesii by RAPDs 1325　
as well as located not far from each other.Thus , ani-
mals activities and seeds spread along the waterway are
likely to have facilitated the gene flow.It also could be
seen from variance analysis of genetic variation that there
was significant differentiation among regions or populations
or individuals within a population (P <0.001).When
strategies for the conservation of germplasm resources of
C.fargesii are implemented , priority should be given to
collect sufficient individuals and the conservation of dif-
ferent populations at the same time should be considered.
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栲树天然群体遗传结构的 RAPD分析
朱其慧　潘惠新　诸葛强　尹佟明　邹惠渝　黄敏仁＊
(南京林业大学林木遗传和基因工程重点实验室 , 南京 210037)
摘要:　利用 RAPD分子标记对 5 个栲树(Castanopsis fargesii Franch.)天然群体共计 188个个体的遗传多样性和群体
遗传结构进行了分析。41 个随机寡核苷酸引物共检测到 385 个位点 , 其中多态位点 157个 ,占 40.78%。物种水平
的Shannon 多样性指数 I=0.459 7 , Nei基因多样度 h=0.296。遗传变异分析表明 , 栲树群体的遗传变异主要存在
于群体内 ,利用 Shannon 多样性指数估算的分化(Hsp_Hpop)/ Hsp=0.047 6 ,遗传分化系数 Gst =0.042 9 , 分子方差分
析(AMOVA)也证实了这一结论 ,群体内的变异组分占了 94.97%, 群体间变异只占 5.03%。 AMOVA 分析结果的显
著性检验也表明 ,群体间及群体内个体间均呈现出显著分化(P<0.001)。
关键词:　栲树;天然群体;RAPD;遗传结构
中图分类号:Q943　　　文献标识码:A　　　文章编号:0577-7496(2002)11-1321-06
收稿日期:2002-02-27　接收日期:2002-08-29
基金项目:国家自然科学基金(39770628)。
＊通讯作者。 (责任编辑:彭　丹(实习))
1326　 植物学报　Acta Botanica Sinica　Vol.44　No.11　2002