全 文 ：Identification of Two Closely-related Species of Eleocharis :
E dulcis and E sphacelata Using RAPD Analysis

Li　Mei
(Department of Agricultural Engineering , Hubei Agricultural College , Jingzhou 434103)
Abstract　Random amplif ied polymorphic DNA (RAPD)was used to identify tw o closely-related
species of E leocharis:E d ulcis and E sphacelata.The use of three decamer primers(SC10-23 , SC10-25
and AB01-18)clustered samples from 8 sites into tw o distinct groups:E dulcis and E sphacelata in a phy-
logenetic dendrog ram obtained by unweighted pair group method analysis(UPGMA)of the genetic dissimi-
larity .RAPD assay proved to be very powerful for distinguishing these two closely related species.
Key words　RAPD;Eleocharis dulcis;E leocharis sphacelata;phy logenetic dendrog ram
The mo rphological differences betw een E dulcis and E sphacelata are that E dulcis comprises a smaller
plant and nut(1.3 ～ 2.1 mm vs.E sphacelata 2 ～ 2.75 mm), has shorter glumes(5 ～ 6.5 mm vs.E
sphacelata 8 ～ 10 mm), and that the stems of E sphacelata are set in close linear series on a stout horizontal
rhizome while those of E dulcis are tuf ted on a slender rhizome[ 1 , 2] .In reali ty , the use of these morpholog-
ical characteristics to separate out the tw o species is not so definitive , especially w hen the w hole plant is not
available.
RAPD(Random Amplified Polymorphic DNA)technique is a polymerase chain reaction(PCR)based
technique which uses short arbit rary oligonucleo tide primers(usually decamer)to di rect DNA polymerase-
mediated amplification of anonymous genetic loci.During past few years , RAPD technique has been used to
estimate genetic relationships w ithin and betw een plant species[ 3] .The objective of this study w as to esti-
mate the discriminatory ability of RAPD markers in classifying tw o closely related species E dulcis and E
sphacelata.
1　Materials and methods
1.1 　Sample collection
Eight si tes(Si te 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8)with w ild E dulcis or E sphacelata w ere sampled in Central
Queensland , Australia.The distance between sites ranged from 2 km to 400 km.Samples f rom Site 1 , 2 ,
5 , 6 and 7 were identified as E dulcis , and Site 8 as E sphacelata by their morphological t raits according to
Sharpe[ 2] .Samples f rom Si te 3 and 4 could not be identified owing to the lack of the w hole plants and the
inflorescences.For most samples , only above-g round stems were collected for RAPD analysis.Another i-
dentified E sphacelata sample(specimen)provided by Leo Duivenvoorden at Central Queensland University
第 18 卷　第 3 期
Vol.18　No.3 　　　　　　　　　
湖　北　农　学　院　学　报
Journal of Hubei Agricultural College
　　　　　　　　1998年 9月
Sept.1998
Received 27 April 1998
This research was supported in part by the funding from RIRDC(Australia)and the CQU URG scheme
The author:Li Mei , female , aged 33 , Master of Science(Biology), Lecturer in the Department o f Agricultural Engi-
neering at Hubei Agricultural College
and a cultivated E dulcis sample w ere included in the analy sis for comparison.
1.2 　DNA extraction
Total genomic DNA was ext racted according to the pro tocol of Doyle &Doyle[ 4] with modifications.
About 0.1 g fresh tissue w as trimmed into a 1.5 mL Eppendo rf tube and g round in 200 μL extract ion
buffer [ 0.1 mol/L Tris-HCl , pH 8.0;0.02 mol/L EDTA;1.4 mol/L NaCl;20 g/L
cetylt rimethy lammonium (CTAB)] w ith a plastic micropestle.Sterile acid-washed sand w as added to aid
g rinding.After grinding , another 300 μL ex traction buffer w as added to the homogenate and mixed w ell
w ith gentle inversion.The homogenate w as centrifuged at 8 000 r/min fo r 5 min.The supernatant(300 ～
400 μL)was transferred to a clean 1.5 mL microfuge tube and emulsified wi th equal volume of chloroform
and isoamy l alcohol (volume ratio 24:1).After cent rifugation at 10 000 r/min fo r 5 min , the aqueous
phase w as recovered.The DNA was precipitated by adding equal volume of ice-cold isopropanol and lef t at
-20 ℃ for at least 30 min.The DNA pellet w as collected af ter cent rifuging for 15 min at 14 000 r/min
and w ashed w ith 400μL volume fraction 70%ice-cold ethanol , vacuum dried , and resuspended in 50μL of
sterile millipore w ater.The DNA was roughly quantif ied by a UV-spect rophotometer at 260 nm o r by run-
ning a 10 g/L agarose gel stained w ith 0.5μg/mL ethidium bromide (EtBr).
1.3 　Oligonucleotide primers
Of fif ty-tw o 10-mer primers screened , three primers (AB01-18:5 -CCACAGCAGT-3 , SC10-23:
5 -GGCTCGTACC-3 and SC10-25:5 -CGGAGAGTAC-3 )that revealed clear polymorphic amplifica-
tion pat terns w ere chosen for the phylogenetic analysis.
1.4　RAPD-PCR
As a result of pilot assays , RAPD-PCR was carried out in a 25 μL reaction containing buffer (10
mmol/L Tris-HCl , pH 8.8 , 50 mmol/L KCl , 1.5 mmol/L MgCl2 , mass fraction 0.1% Triton X-100),
200 μmol/ L of each deoxynucleotide t riphosphate (dNTP———ATP , dG TP , dCTP , dTTP), 0.2μmol/L
primer , 0.5 U Red-Hot Taq polymerase(Advanced Bio technologies L td), and 20 ～ 80 ng DNA ex tract.
This mix ture w as overlaid w ith 10μL mineral oil(Sigma Chemical Co).The DNA amplification was per-
formed in a M inicyclerTM(MJ Research)prog rammed fo r one cycle at 94 ℃ for 2 min , followed by 39 cy-
cles at 94 ℃ for 1 min , 33 ℃for 1 min and 72 ℃for 50 s and a f inal cy cle at 72 ℃ for 1.5 min.A nega-
tive control consisting of all reaction components except genomic DNA was included in each set of amplifica-
tion.
The amplified products(10 ～ 12μL)mixed w ith 5μL loading buf fer(2.5 g/L bromphenol blue , 400
g/L sucrose)were elect rophoresed on a 12 g/L DNA grade agarose (Sigma Chemical Co)gel containing
0.5 μg/mL EtBr in 1×TAE buffer at 100 V for 65 min.A 123-bp DNA ladder(Sigma Chemical Co)was
used as a relative molecular mass marker.After elect rophoresis , the gel w as further stained by soaking in
0.5 μg/mL EtBr for 20 min and destained under running w ater for 10 min.RAPD profiles were visualised
and pho tographed under a UV transilluminator using Polaroid 665 instant film.The RAPD bands w ere
scored f rom both the pho tographs and the film negatives.
1.5　Data analysis
RAPD bands w ere scored as present ” 1” or absent ” 0” .A matrix of dissimilarity w as generated by
RAPDPLOT[ 5 , 6] using Nei and Li s[ 7] similarity index
　　S =2 NAB/(NA + NB)
where NAB is the number of common bands shared by individuals A and B , NA is the number of bands
in individual A and N B is the number of bands in individual B.The genetic dissimilarity betw een A and B is
1-S .The dissimilarity matrix was entered into the N EIGHBOR prog ram on PHYLIP 3.5 C[ 8] .The den-
214 湖　北　农　学　院　学　报　　　　　　　　　　　　　　　1998年
drog ram was produced w ith Unw eighted Pair Group Method Analysis (UPGMA) and plotted using
DRAWGRAM on PHYLIP 3.5 C.
2　Results
2.1　DNA extraction and RAPD-PCR
The yields of DNA varied g reatly , ranging f rom 80 to 1 500 μg/g for fresh material w hen measured
w ith a UV spectropho tometer.The ex tracted DNA was alw ays accompanied by brown or black coloured
compounds , which sometimes totally inhibited RAPD amplification.Shearing (i.e.a smear of low relative
molecular mass f ragments)of DNA was observed from time to time.Removing the brow n pellet when
w ashing the DNA w ith volume fraction 70%ethanol resulted in quite a few DNA extracts that were ampli-
f iable , and which otherwise could not give any RAPD bands.
2.2　 Identi fication of E dulcis and E sphacelata by RAPDs
Samples f rom 8 sites as w ell as one identified E sphacelata sample and one cult ivated sample (code:
GT)were amplif ied w ith three primers:primer AB01-18 , SC10-23 , SC10-25.Figure 1 presents the
RAPD profile amplified wi th primer SC10-25.From these results , a dissimilarity matrix and a phylogenet ic
dendrog ram were generated.As seen from Figure 2 , these three primers successfully grouped the samples
from all sites into tw o groups:E dulcis and E sphacelata .Samples f rom Site 1 , 2 , 3 , 4 , 5 , 6 and 7 to-
gether with the cultivated E dulcis sample were placed into one group.The sample from Site 8 and the i-
dentified E sphacelata were placed , based on a 37% genetic dissimilarity index , into the o ther group as ex-
pected.The genetic dissimilarity betw een wild E dulcis ranged from 0%(Site 1 , 2 , 3 and 4)to 45.5%
(betw een Site 6 , 7 and Si te 1 , 2 , 3 , 4 , 5)and from 45.5% to 67% between cultivated and w ild E
dulcis.
Figure 1 　RAPD profile of wild and cultivated E dulcis
samples, and E sphacelata samples amplif ied by primer
SC10-25.Lanes f rom left t o right are:lane 1—DNA marker(123-
bp DNA ladder);lane 2—GT (cultivated E dulcis);lane 3—Site 6;
lane 4—Esp(ident ified E sphacelata specimen);lane 5—S ite 7;lane
6—Site 8;lane 7—Si te 5;lane 8—Site 3;lane 9—Site 4;lane 10—
Site 2;lane 11—Site 1;lane 12—negative control.
Figure 2 　Phylogenetic dendrogram of wild and culti-
vated E dulcis samples , and E sphacelata samples con-
structed with 30 RAPD markers(3 primers), based on
UPGMA analysis using similarity index of Nei and
Li[ 7] .Site 1～ 8 refers to 8 sampling sites.Espha is an ident i-
fied E sphacelata specimen and GT is a cult ivated E dulci s sam-
ple.
215第 3 期　Li Mei:Identif icat ion of Tw o Closely-related Species of Eleocharis:E dulcis Zand E sphacelata Using RAPD Analysis
3　Discussion
3.1　DNA extraction and RAPD-PCR
The major concern about DNA extraction is the quanti ty and quality of DNA recovered.Yields of
crude DNA from 80 to 1 500 μg/g for fresh material w ere reco rded in the present study.However , the
measurement based on the absorbance at 260 nm may not be a reliable indication of DNA concentration ow-
ing to the presence of RNA and other contaminants in DNA ex tracts.High DNA yields are not necessarily
associated with the suitability fo r RAPD amplification.In fact , nanogram quanti ties of DNA are sufficient
for RAPD amplification and DNA yield is therefo re not a very important consideration.In contrast , DNA
quality plays a more impo rtant role in RAPD amplification.
One indication of DNA quality is the size of prepared DNA.Generally speaking , macro-molecular DNA
(50 ～ 100 kb in leng th)and wi thout obvious shearing is preferred for subsequent molecular analysis[ 9] , al-
though it may not be necessary for RAPD amplification[ 10] .DNA shearing may be caused by DNA deg rada-
tion and/or mechanical shearing during sample preservation and cell disruption[ 9 , 11] .DNA isolated from
stems of some E dulcis and the E sphacelata specimen show ed smearing indicating that the DNA had been
sheared.However , the shearing of the DNA did not show obvious effects on RAPD amplification.
The other indicat ion of DNA quality is the presence or absence and the amount of PCR inhibi tors such
as polyphenolics and polysaccharides , which are mainly determined by plant species[ 4 , 9 , 11～ 13] .As for
many plant species , polyphenolics seemed to be the main source of PCR inhibito rs in DNA extracts of E
dulcis and E sphacelata .High amount of polyphenolics w as evidenced by highly brow n or black coloured
compounds copurified wi th DNA pellets.Phenols are known to bind to pro teins by forming hydrogen bonds
w ith peptide bond oxygens[ 14] .The phenolic compounds can sometimes totally inhibit PCR reaction.Tak-
ing out the brow n pellets w hen w ashing w ith volume fraction 70% ethanol enabled acquisition of RAPD-
amplifiable DNA , but the DNA concentration may be reduced to a very low level , as most DNA was sup-
posed to exist in the brow n pellets.
3.2　 Identi fication of E dulcis and E sphacelata by RAPDs
Previous studies have suggested that RAPD analy sis is a reliable technique fo r estimating relationships
at the species or lower level[ 3 , 15～ 17] , although the utility of RAPD analysis for higher taxonomic levels is
still a mat ter of cont roversy due to the uncertainty of the homologous nature of RAPD bands with similar
size[ 15 , 18] .The use of three primers in this study clustered samples f rom all si tes into tw o groups:E dulcis
and E sphacelata .This result is consistent w ith the identification based on morpholog ical characters.
The int raspecific dissimilarity w as remarkably variable , rang ing f rom 0% to 45.5%fo r w ild E dulcis.
The geog raphic isolation may no t be the main reason for the relatively high int raspecific dissimilarity , since
samples from Site 4 and Site 1 , 2 and 3 exhibited identical RAPD profiles although they are separated by
considerable distance(more than 300 km from Site 4 to Site 1 , 2 and 3).Small sample size and small num-
ber of RAPD markers may have resulted in the overestimation of intraspecific dissimilari ty.The origin of
diversi ty existing in E dulcis may have resulted f rom inter-or intra-specif ic hybridization and variants dis-
tributed by birds and o ther animals.Random mutation accumulated over a long time may also contribute to
the genetic diversity in this species.
Acknowledgments:This work is part of the author s Master project under taken at Central Queensland University , Aus-
tralia.The guidance from Pro f.David M idmore and Dr.Patrick Whitty is gratefully acknow ledged.
216 湖　北　农　学　院　学　报　　　　　　　　　　　　　　　1998年
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用 RAPD技术区分荸荠属中亲缘关系相近的两个种:
Eleocharis dulcis和 Eleocharis sphacelata
李　梅
(湖北农学院农业工程系 , 荆州 434103)
摘　要 　用 RAPD 技术鉴别了荸荠属中亲缘关系相近的两个种:E dulcis 和 E
sphacelata 。根据 3个 10碱基随机引物(SC10-23 ,SC10-25和 AB01-18)获得的遗传距离值 ,用
UPGMA 聚类方法构建了来自 8个样点的样本的分子系统树。在分子系统树中 ,这 8 个样点
的样本被分为两组:E dulcis和E sphacelata 。
关键词　随机扩增多态性 DNA(RAPD);荸荠;分子系统树
中图法分类号　Q949.71
217第 3 期　Li Mei:Identif icat ion of Tw o Closely-related Species of Eleocharis:E dulcis Zand E sphacelata Using RAPD Analysis