全 文 ：应用 RAPD技术研究阿尔卑斯山黄花茅
居群内的遗传分化
赵 桂 仿 　Francois Felber　Philippe Kuepfer
(Botanical Institute , University of Neuchatel , Chantemerle 22, CH-2007 , Neuchatel , Switzerland)
Subpopulation differentiation of Anthoxanthum alpinum
(Poaceae)along an altitudinal gradient detected
by random amplified polymorphic DNA
ZHAO Gui-Fang＊　Francois Felber　Philippe Kuepfer
(Botanical Institute , University of Neuchatel , Chantemerle 22, CH-2007 , Neuchatel , Switzerland)
Abstract　Random amplified polymorphic DNA(RAPD)phenotypes generated by 15 primers were
scored in 5 subpopulations of Anthoxanthum alpinum along an altitudinal gradient.Although few
subpopulation-specific markers were found , a significant population differentiation was revealed by
the use of principal factor analysis and Ward s cluster analysis.The result was also confirmed by
the significant positive correlation when comparing both the matrix of similarity between the individu-
als within subpopulations and the matrix among subpopulations , as well as the matrix of distances
and the differences of altitude between the subpopulations(Mantel test).This study suggests that
the differences of altitude between subpopulation sites may result in the differences of phenological
period for flowering and growth which restrict gene flow.
Key words　Anthoxanthum alpinum;Genetic differentiation;RAPD;Altitudinal gradient
Genetic differentiation of natural populations is considered as a dynamic process depending on
the balances between gene flow and selection.Gene flow acts as a unifying factor.A substantial
gene flow can hamper genetic differentiation.Selection which favors adapted genotypes in a heterozy-
geneous environments can override the homogenizing forces of gene flow , even when gene flow occurs
(Caisse &Antonovics , 1978;Endler , 1977;Antonovics et al., 1971).
Altitudinal gradients have been favoured for the study of ecotypic differentiation for a long time
(Oyama et al., 1993;Clausen et al., 1940).The advantage of elevational transects is that rapid
environmental changes often occur over short distance , e.g.the temperature decreases and the pre-
cipitation increases generally with the increase of altitude (Ozenda , 1985).Moreover , the differ-
ences of altitude could result in the changes in flowering onset and the period for growth , thus re-
stricting gene flow between subpopulations of different altitudes (Galen &Kevan , 1980;Waser ,
1978).
Recently , random amplified polymorphic DNA (RAPD)markers have become increasingly
popular in their applications to population biology in spite of their properties of dominance.The ad-
vantages of this technique over conventional molecular markers such as RFLP are that large numbers
of samples can be analyzed economically and quickly , that only minimal-quantites of material is
Present address:Department of Biology , Northwest University , Xi an 710069 ,Shaanxi , China
1998-08-26收稿 ,1999-04-16收最后修改稿。
植 物 分 类 学 报　38(1):64 ～ 70(2000)
Acta Phytotaxonomica Sinica
needed for each sample , that the specific DNA fingerprints obtained are independent of ontogenic
expression , and that most of the genomes can be sampled with a potentially unlimited number of
markers.RAPD markers have been used to identify different individual genotypes and species(Liu
&Furnier , 1993;Halward et al., 1991), to study mating system in natural populations(Arnold et
al., 1991), to assess patterns of paternity and kinship(Lewis &Snow , 1992), and to detect and
analyse genetic variation (Castagnone-Sereno et al., 1994;Dawson et al., 1993;Williams et
al., 1990).
In this study , we report the use of RAPD markers to assess genetic differentiation between the
subpopulations of A.alpinum along an altitudinal gradient.
1　Materials and methods
1.1　Plant materials
Anthoxanthum alpinum Löve &Löve (Poaceae)is a perennial grass.Individuals of this
species are diploid , predominantly outcrossing (Felber , unpublished data;Zeroual-Humbert-Droz ,
1995).In the Alps , A.alpinum covers a considerable altitude range because it grows throughout
the subalpine and alpine zones above 1700 m (Felber , 1986;Favarger , 1962).At lower eleva-
tions , A.alpinum is replaced by a closely related species , A.odoratum (Felber , 1988 , 1986).
Sampling was performed at Belalp of the Swiss Alps.It is located in the southern part of the
Aar massif of the Aletsch region on the right side of the Rhone valley.An altitudinal gradient was
set up on an eastern slope , located on the versant of the Hofathorn between 2020m and 2830 m with
a distance of 1.75 km.
Subpopulations were sampled every 200 m altitude intervals.In 1993 , four subpopulations
were collected and sampling was completed in 1994 with an additional subpopulation (2830 m)in
order to cover all altitudes.Each subpopulation consisted of 50 ～ 80 individuals collected at a dis-
tance of at least one-meter.The collected plants were then cultivated in the experimental garden of
the University of Neuchatel.
1.2　DNA isolation and polymerase chain reaction(PCR)
Experiments were conducted in 1995.Five individuals were randomly chosen for each subpop-
ulation.Total genomic DNA was isolated from fresh leaf material of single plant growing in the ex-
perimental garden by using the standard CTAB protocol (Doyle &Doyle , 1987).PCR was per-
formed in volumes of 25 μl consisting of about 10 ng of genomic DNA , 1.5 μM primer (Operon
Technologies Primer OPB and OPP series:no.1 ～ 20.), 1 ×Goldstar (polymerase)reaction
buffer , 1.5 mM MgCl , 2.5 mM each of dATP , dCTP , dGTP and dTTP , 0.625 unit of Goldstar
DNA polymerase.The thermal cycle was set for 1 cycle at 94℃ for 3 min.followed by 35 cycles of
30 sec.at 94℃, 45 sec.at 36℃ and 1 min.and 30 sec.at 72℃.and completed by one cycle of
7 min.at 72℃.Five μl reactionmix of each sample was loaded and run on minigel(1.6%agarose
in TBE buffer containing ethidium bromide)and visualized under UV-light.
1.3　Data analysis
djk=[ ∑P
i=1(xij-xik)2]
1
2
Ward s cluster analysis and principal factor analysis , by the use of computer program
STATISTA , were made to get an overview of the genetic variation and differentiation in the materials
studied.Euclidean distance in the cluster analysis is defined as a measure of variance between two
entities , j and k .In the principal factor analysis , factor 1 (axis 1)described 35% of variation ,
factor 2(axis 2)decribed 10% of variation , and factor 3 (axis 3)described 7% of variation.In
order to estimate the level of significance of genetic differentiation between the subpopulations and
the correlation between the genetic differentiation and geographical distance (altitudinal gradients
1 期 赵桂仿等:应用 RAPD技术研究阿尔卑斯山黄花茅居群内的遗传分化 65　
therein), we tested the correlation between matrix of similarity of individuals within subpopulations
and compared the matrix of distances between the paired subpopulations with the differences of alti-
tude by a Mantel test.999 permutations were performed.
2　Results
Forty primers of OPB andOPP series were used for an initial screening.Finally , fifteen of the
OPB series , which generated informative amplification products , were chosen for the whole experi-
ment.The information of RAPD primers used in the analysis of genetic variation in this study , the
mean number of products per subpopulation and the number of unique products are presented in
Table 1.
Table 1　RAPD primers used in the analysis of variation in 5 subpopulations of A.alpinum along
an altitudinal gradient.
Primer code
Sequence
(5′to 3′)
Mean number of products
per subpopulation
No.unique
bands
2020 2200 2405 2575 2830
OPB-01 GTTTCGCTCC 6.8 4.8 8.0 8.0 10.0 4
OPB-02 TGATCCCTGG 5.2 5.8 4.4 4.6 6.4 0
OPB-03 CATCCCCCTG 5.4 6.6 4.8 5.2 5.6 0
OPB-04 GGACTGGAGT 6.0 5.4 3.4 4.0 4.6 1
OPB-05 TGCGCCCTTC 9.2 10.8 9.2 7.8 10.2 0
OPB-07 GGTGACGCAG 10.6 10.0 7.4 10.0 10.2 1
OPB-08 GTCCACACGG 4.6 4.4 4.6 4.2 5.6 1
OPB-09 TGGGGGACTC 4.8 5.4 5.2 5.8 4.4 1
OPB-10 CTGCTGGGAC 4.6 3.6 4.4 4.0 4.2 1
OPB-11 GTAGACCCGT 5.8 6.4 5.6 4.8 5.6 0
OPB-12 CCTTGACGCA 5.8 6.4 4.8 5.8 6.6 0
OPB-13 TTCCCCCGCT 5.4 4.8 4.8 4.2 4.2 0
OPB-14 TCCGCTCTGG 3.4 3.2 3.6 4.0 3.6 0
OPB-15 GGAGGGTGTT 4.0 4.0 5.2 5.0 5.4 0
OPB-16 TTTGCCCGGA 3.4 3.0 3.6 3.6 4.4 0
Mean 5.7 5.6 5.3 5.4 6.1
Considerable variation was found within subpopulations in this study , and only few subpopula-
tion-specific RAPD markers from 15 variable oligonucleotide primers were identified for 25 individu-
als from 5 subpopulations (Table 1).However , a tree diagram from Ward s analysis of cluster
showed that 25 individuals were clearly divided into three larger groups while linkage distance was
7.6.In the diagram , the highest subpopulation (2830 m)was separated from the others(Fig.1 ,
the middle of diagram), and the other two groups were further divided into two smaller groups , re-
spectively .The result suggested that individuals of the same subpopulation had similar genetic vari-
ability , while the 5 subpopulations were separated from each other.These results were confirmed by
the analysis of Gower s(1985)similarity between the individuals of the same subpopulation(R=
0.59 , P<0.001).In addition , an analysis of principal factors also showed three similar groups ,
with the highest subpopulation(individuals 21 ～ 25)being clearly separated from the others(Fig.
2).By Mantel test , we transformed the matrix of similarity into Jaccard s(1908)matrix of distance
and analysed the correlation between the matrix of distance and the differences of altitude.The re-
sults also revealed a significant positive correlation between the genetic distance and altitude (R=
0.64 , P<0.001 and 999 permutations were used).Moreover , the analysis of paired matrix of dis-
66　 植　物　分　类　学　报 38 卷
tance between the subpopulations showed that genetic differentiation between the subpopulations a-
long altitudinal gradient was significant(P<0.05)except for the subpopulations 2020 ～ 2200 and
2405 ～ 2575(P=0.144)(Table 2), because these two subpopulations were similar in genetic vari-
ability and were in an identical group in the above analysis(see Fig.1 and Fig.2).
Fig.1　Tree Diagram for 25 Cases;Ward s method;Euclidean distance
Fig.2　Factor Loadings , Factor 1;Unrotated;Principal factors(Cent roid)
1 期 赵桂仿等:应用 RAPD技术研究阿尔卑斯山黄花茅居群内的遗传分化 67　
Table 2　Pairwise analysis of distance matrix of RAPD and significant level(P)measured by Mantel test.
pairs of subpopulations P
2020-2200 ns
2020-2405 ＊
2020-2575 ＊
2020-2830 ＊
2200-2405 ＊
2200-2575 ＊
2200-2830 ＊
2405-2575 ns
2405-2830 ＊
2575-2830 ＊
Note:ns———no signifi cant;＊———P < 0.05
3　Discussion
Genetic differentiation for morphological and allozymic , as well as DNA characters in natural
plant populations along a geographic distance is a common observation (Dawson et al., 1993;
Saghai Maroof et al., 1990;Mitton et al., 1980;Bergmann , 1978;Tigerstedt , 1974;Clegg &
Allard , 1972).Traditional views assumed that there would be little genetic differentiation within
continuous populations(Mayr , 1963), but , more recent observations illustrated that genetic differ-
entiation does occur over short distances in many plants(Galen et al.,1991;Bos et al., 1986;
Jain &Bradshaw , 1966;Grant&Hunter , 1962)as the result either of spatial variation in heteroge-
neous environments or of local gene flow (Brown , 1979;Endler , 1977;Levin an Kerster , 1974;
Bradshaw , 1972).Higher levels of gene flow can counteract the action of selection by reducing ge-
netic differentiation among populations(Jain &Bradshaw , 1966).Restricted gene flow may permit
local adaptation(Slatkin , 1985;Dickinson &Antonovics , 1973;Jain &Bradshaw , 1966)and can
foster genetic isolation by distance (Endler , 1977;Wright , 1951).
Despite the presence of few genetically identical RAPD genotypes within subpopulations , which
indicates that A.alpinum is a highly outcrossing species with a corresponding high degree of genetic
variation , a significant subpopulation differentiation within the transect was revealed using RAPD
markers.A good agreement was obtained from the results of the different statistical methods used for
analysing population structure in this study.
In our study sites , A.alpinum grows in a continuous population along the altitude gradient.
However , genetic differentiation was not found to be random as a significant correlation was observed
in this study , which suggests that the differences of altitude between subpopulation sites may result
in the differences of phenological period for flowering and growth which restrict gene flow.
Acknowledgements　This study was supported by the Swiss National Foundation for Scientific Research (Grant
50001-35250)and is part of a Ph.D.thesis of G.F.Zhao presented at the University of Neuchatel.
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摘要　应用 RAPD技术 , 沿一个海拔梯度研究了阿尔卑斯山黄花茅自然居群的遗传变异和分化。实验
表明 , 虽然在亚居群间有很少的亚居群独有遗传标记的存在 , 但通过 RAPD资料的聚类分析 、主因子分
析以及相关分析观察到遗传分化沿海拔梯度发生 ,而且亚居群间的遗传分化和它们的海拔高度(地理距
离)呈有意义的正相关。研究结果暗示 , 在阿尔卑斯山的高山-亚高山过渡区 , 亚居群间的海拔高度差别
可能导致黄花茅开花和生长物候期的变化 ,而后者限制了亚居群间的基因流 ,从而引起居群内的遗传分
化。
关键词　黄花茅;遗传分化;RAPD;海拔梯度
70　 植　物　分　类　学　报 38 卷