Nelumbo nucifera using nuclear ribosomal ITS, ISSR RAPD markers

Analyses of genetic relationships in Nelumbo nucifera using nuclearribosomal ITS sequence data,ISSR and RAPD markersYan-Chuang Han a ,Cai-Zhu Teng c ,Fu-Haosen Chang a ,Gituru W.Robert d ,Ming-Quan Zhou b ,*,Zhong-Li Hu a ,b ,**,Yun-Chun Song aaKey Lab of the Ministry of Education for Plant Developmental Biology,College of Life Science,Wuhan University,Wuhan 430072,ChinabLotus Center,Wuhan University,Wuhan 430072,ChinacCollege of Life Science,Guangxi Normal University,Guilin 541004,ChinadBotany Department,Jomo Kenyatta University of Agriculture and Technology,Nairobi 63000-00200,KenyaReceived 24June 2006;received in revised form 22March 2007;accepted 16April 2007Available online 20April 2007AbstractDespite the economic importance of Nelumbo nucifera ,there have been no molecular studies on genetic relationships among cultivars in the species.In the present study 38accessions were sampled including 37accessions of N.nucifera or hybrids between N.nucifera and Nelumbo lutea and a single accession of N.lutea .In the ITS analyses,Chinese and Japanese lotus comprise a single cluster with a moderate bootstrap support 68%indicating there is very high similarity between them.Moreover,these ISSR and RAPD results also indicate that there is very close genetic relationship between Chinese and Japanese lotus.In the ISSR and RAPD analyses,although 38accessions all are distinctly separately into two groups,viz.N.nucifera and N.lutea ,there is a high Jaccard similarity coefficient (0.785and 0.656)between the two species.In N.nucifera the two different groups of the species,viz.flower lotus and rhizome lotus accessions show clear genetic variations.Seed lotus accessions do not form a distinct cluster but are interspersed among the flower accessions indicating that seed lotus is phylogenetically close to flower lotus and they might originate from close wild lotus in genetic relationship.In flower lotus,big-flower type accessions and medium-small type accessions have obvious genetic variation,indicating height is an important criterion in the classification system of flower lotus.#2007Published by Elsevier B.V .Keywords:Genetic relationship;ISSR;ITS;Nelumbo lutea ;Nelumbo nucifera ;RAPD1.IntroductionThe Nelumbonaceae also known as the lotus family is a small family of perennial,aquatic angiosperms which traditionarily consists of the two species Nelumbo nucifera Gaertn.and Nelumbo lutea (Willd.)Pers.based on the morphological characters.The family is characterized by simple,peltate leaves which lack stipules and are borne on the surface of the water.N.nucifera ,also called the Indian or sacred lotus including wild lotus and a number of cultivars,is found throughout Asia and Australia,whereas N.lutea ,also known as the American lotus or water chinquapin occurs in eastern andsouthern North America.The lotus is an important aquatic economic plant,not only as a dainty and ornamental flower but also as a source of herbal medicine with strong antipyretic,cooling,astringent,and demulcent properties (Mukherjee et al.,1997;Sinha et al.,2000;Qian,2002).More recently,evidences from reproductive biology studies,pollen and anther studies,cytogenetics and rbc L sequence studies,suggest that there is very high similarity between the two Nelumbo species,thus N.lutea has been considered to be a subspecies of N.nucifera (Les et al.,1991;Huang et al.,1992;Borsch and Barthlott,1994;Wang et al.,1997;Sarah and Jeffrey,1999;Diao et al.,2005).To date no molecular analysis has been performed to test genetic relationships of accessions in the species N.nucifera .ITS has been widely used in phylogenetic and phylogeo-graphical studies on a variety of organisms (Baldwin et al.,1995),and it possesses features that allow addressing phylogenetic questions at interspecific level within a genus (Baldwin,1993;Ritland et al.,1993;Soltis and Kuzoff,1993).ISSR and RAPD markers have been successfully used for DNA/locate/aquabotAquatic Botany 87(2007)141–146*Corresponding author at:Lotus Center,Wuhan University,Wuhan 430072,China.Tel.:+862768753606;fax:+862787862967.**Corresponding author at:Key Lab of the Ministry of Education for Plant Developmental Biology,College of Life Science,Wuhan University,Wuhan 430072,China.Tel.:+862768753606;fax:+862787862967.E-mail addresses:loyjzx@ (M.-Q.Zhou),huzhongli@ (Z.-L.Hu).fingerprinting (Moreno et al.,1998;Blair et al.,1999;Divaret et al.,1999;Gilbert et al.,1999),for population genetic studies (Wolfe et al.,1998;Nebauer et al.,1999;Li et al.,2005)and phylogenetic studies (Hess et al.,2000;Mattioni et al.,2002).So far,only two studies have been made to characterize the genetic relationships between ancient and modern lotus and genetic diversity in wild lotus in the species by using RAPD and ISSR (Zou et al.,1998;Han et al.,2007).The present study was conducted to determine the genetic variability in N.nucifera using ITS,ISSR and RAPD markers.Our aims were to:(1)assess genetic relationships among accessions in N.nucifera ;(2)obtain genetic relationships between Chinese and Japanese lotus.2.Materials and methods 2.1.Plant materialsThirty-eight accessions of Nelumbo were used in the genetic analyses.Among them,there are 33accessions of N.nucifera ,1accession of N.lutea as an out-group and 4accessions of their hybrids.Accessions codes,types,sources and origins are provided in Table 1.About 5g of young leaves from each accession were obtained and placed in a ziplock plastic bag containing about 50g of silica gel which speeded up the drying process.The samples were stored at room temperature in the laboratory.In order to further test the genetic differentiations between Chinese and Japanese accessions,six typical accessions were sampled.Among them,there are two Chinese accessions,N.nucifera cv.‘Taikong’and N.nucifera cv.‘Chinese Antique Lotus’;three Japanese accessions,N.nucifera cv.‘Ohga Lotus’,N.nucifera cv.‘Sunyatsen Lotus’and one hybrid of the two species—N.nucifera cv.‘Wufeilian’;one accession of N.lutea .Six ITS sequences were sequenced.All sequences were submitted to GenBank,whose GenBank accession numbers are DQ 105981to DQ 105985and AY 858639,respectively.Ceratophyllum demersum is used as the out-group.Table 1Thirty-eight Nelumbo accessions used in the present study (LCWU:Lotus Center,Wuhan University;NHFG:Nanjing Horticulture Flower Garden;WVSGS:Wuhan Vegetable Scientific Graduate School)Codes Accesions nameType Sources Origins 1Nelumbo nucifera cv.‘Taikong’Seed lotus WVSGS China 2N.nucifera cv.‘Hubei-20Rhizome lotus WVSGS China 3N.nucifera cv.‘Hubei-30Rhizome lotus WVSGS China 4N.nucifera cv.‘Hubei-40Rhizome lotus WVSGS China 5N.nucifera cv.‘Hubei-50Rhizome lotus WVSGS China 6N.nucifera cv.‘Eouza’Rhizome lotus WVSGS China 7N.nucifera cv.‘New One’Rhizome lotus WVSGS China 8N.nucifera cv.‘92170Rhizome lotus WVSGS China 9N.nucifera cv.‘Chinese Antique Lotus’Flower lotus LCWU China 10N.nucifera cv.‘Ohga Lotus’Flower lotus LCWU Japan 11Nelumbo lutea (Willd.)Pers.Flower lotus LCWU America 12N.nucifera cv.‘Huangwufei’Flower lotus NHFG China 13N.nucifera cv.‘Wufeilian’Flower lotus LCWU Japan 14N.nucifera cv.‘Wecoming Guests’Flower lotus LCWU China 15N.nucifera cv.‘White Cherry’Flower lotus LCWU China 16N.nucifera cv.‘Sunyatsen Lotus’Flower lotus LCWU Japan 17N.nucifera cv.‘Gongxunlian’Flower lotus LCWU China 18N.nucifera cv.‘Ziyihongshang’Flower lotus LCWU China 19N.nucifera cv.‘Taohongsuyu’Flower lotus LCWU China 20N.nucifera cv.‘Yanhongzhuang’Flower lotus LCWU China 21N.nucifera cv.‘Chutianxiaoxia’Flower lotus LCWU China 22N.nucifera cv.‘Jingzhi-10Flower lotus LCWU China 23N.nucifera cv.‘Jingzhi-20Flower lotus LCWU China 24N.nucifera cv.‘Hunan’Seed lotus LCWU China 25N.nucifera cv.‘Yinhongqianye’Flower lotus LCWU China 26N.nucifera cv.‘Meizhise’Flower lotus LCWU China 27N.nucifera cv.‘Pink Lotus’Flower lotus LCWU China 28N.nucifera cv.‘Xuanwuhu Red’Flower lotus NHFG China 29N.nucifera cv.‘Birthday’s Peach’Flower lotus NHFG China 30N.nucifera cv.‘Hongxituozhu’Flower lotus NHFG China 31N.nucifera cv.‘Changing Face’Flower lotus NHFG China 32N.nucifera cv.‘Big Versicolor’Flower lotus NHFG China 33N.nucifera cv.‘Pink Double Palace’Flower lotus NHFG China 34N.nucifera cv.‘Shaoxing Pink’Flower lotus LCWU China 35N.nucifera cv.‘Sino-Japanese Friendship’Flower lotus LCWU China 36N.nucifera cv.‘Taibailian’Flower lotus LCWU Japan Y.-C.Han et al./Aquatic Botany 87(2007)141–1461422.2.DNA extractionGenomic DNA of every sample was isolated according to the CTAB protocol of Doyle and Doyle(Doyle and Doyle,1987).2.3.ITS PCR amplification,cloning,and sequencingThe ITS region,including ITS1,the5.8S rRNA gene,and ITS2,was amplified by PCR using the universal primer pair ITS5(50-CGAAGTAAAAGTCGTAACAAGG-30)and ITS4 (50-TCCTCCGCTTATTGATATGC-30).Each25m L PCR mix-ture contained:50ng of total genomic DNA,400mM each dNTP,1U Taq polymerase(Promega,Madison,USA), 1ÂTriton-X PCR buffer, 2.0mM MgCl2,and0.4mM of each primer.PCR amplifications on a PÂ2Thermal Cycler (Thermo Electron Corporation,Madison,USA)followed a given protocol:5min at948C;40cycles of1min at948C, 1min508C and2min728C;7min at728C andfinal cooling to48C.The interesting bands were excised from a2%agarose gel in 0.5ÂTBE buffer and purified using a gel extraction kit (TakaRa Biotechnology Co.Ltd.,Dalian,China).PCR products were cloned using a pGEMT-easy vector system(Promega,Madison,USA).A total of500ng of purified plasmid DNA was then sequenced in both directions on an ABI3730XL sequencer (Sunbiotech Co.Ltd.,Beijing,China).All sequences were submitted to GenBank.2.4.Evaluation of primersA total of60ISSR primers(AuGCT Co.Ltd.,Beijing, China)were screened initially screened infive selected samples from the38accessions.After assessing the effects of Mg2+ concentration,template DNA concentration,and temperature during the annealing stage of amplification,we selected13 primers that produced clear and reproducible fragments for further analyses(Table2).Because RAPD is sensitive to PCR reaction parameters,85 primers(AuGCT Co.Ltd.,Beijing,China)were screened with all the accessions.The effects of Mg2+and template DNA concentrations were examined.Under the optimized condition,54out of85primers generated strong amplified products.Thereafter a subset of19primers which produced clear and reproducible bands was selected for further analyses (Table2).Table2Primers used in RAPD and ISSR amplifications(B,C/G/T;D,A/G/T;H,A/C/T;V,A/C/G;Y,C/G)Primer type Sequence(50-30)Annealing temperature(8C)No.of recorded bands No.of polymorphism bands RAPD primersOPL02TGGGCGTCAA35109OPM02ACAACGCCTC351311SBS A11CAATCGCCGT351210SBS A13CAGCACCCAC351310SBS A14TCTGTGCTGG3597SBS A15TTCCGAACCC351411SBS A18AGGTGACCGT35108SBS B01GTTTCGCTCC3554SBS B03CATCCCCCTG35108SBS B04GGACTGGAGT351312SBS B06TGCTCTGCCC3597SBS C08TGGACCGGTG351311SBS C09CTCACCGTCC351212SBS C12TGTCATCCCC351311SBS C13AAGCCTCGTC351010SBS C14TGCGTGCTTG351211SBS D11AGCGCCATTG351312SBS D13GGGGTGACGA351210SBS D14CTTCCCCAAG3597ISSR primersUBC810(GA)8T55119UBC811(GA)8C551510UBC823(TC)8C551211UBC834(AG)8YT52109UBC861(ACC)55865UBC862(AGC)5581412UBC864(ATG)558149UBC866(CTC)558105UBC873(GACA)4562116UBC880(GGAGA)358106UBC889DBD(AC)7551412Y.-C.Han et al./Aquatic Botany87(2007)141–1461432.5.ISSR amplificationISSR amplification was performed according to the protocol of Han et al.(2007).PCR products were analyzed on 2.0%agarose gels in 0.5ÂTBE buffer at a constant voltage of 100V approximately 4h,then stained with ethidium bromide,visualized with ultraviolet light and photographed.2.6.RAPD amplificationRAPD amplification with nineteen primers was repeated two times.The amplification was performed according to the protocol of Li et al.(2005).PCR products were analyzed on 1.5%agarose gels at a constant voltage of 100Vapproximately 3h,then visualized with ultraviolet light and photographed.2.7.Data analysesSequences were aligned using CLUSTALW (Thormann et al.,1994).Phylogenetic analyses of the complete data set of sequences were conducted using the test version of PAUP4.0b10(Swofford,2001),and heuristic searches were done with COLLAPSE,MULPARS and TBR branch-swapping options to save all of the equally most parsimonious trees.All characters were of the type ‘‘unordered’’and had equal weight.Gaps were treated as ‘‘missing’’.Bootstrap analyses of 1000replications were performed to show relative support for individual clades.Both ISSR and RAPD are dominant markers.Their amplified fragments were scored for band presence (1)or absence (0)and two binary qualitative data matrices were constructed.Data analyses were performed by using the NTSYS-pc version 2.1computer program package (Rohlf,2000).Pair-wise comparisons were calculated using the Jaccard similarity coefficient (Kosman and Leonard,2005).The similarity values were used to generate a dendrogram via the un-weighted pair group method with arithmetic average (UPGMA).3.ResultsITS1and ITS2sequences with an aligned length of 505base pairs were obtained for the six accessions.Of these 505base pairs,226(27%)were constant characters;266(53%)were parsimony-uninformative variable characters;13(2%)were parsimony-informative characters across all the six accessions.Parsimony analyses of the data yielded the strict consensus of seven most parsimonious trees with a consistency index (CI)of 0.967,retention index (RI)of 0.412,homoplasy index (HI)of 0.033,and rescaled consistency index (RC)of 0.398.Chinese and Japanese lotus comprise a single group (68%bootstrap support),and N.lutea and N.nucifera cv.‘Wufeilian’cluster together (72%bootstrap support).Thirteen selected ISSR primers generated a total of 164fragments including 126polymorphic bands (76.83%),on average of 12.62bands per primer (Table 2).Amplification of genomic DNA of38Y.-C.Han et al./Aquatic Botany 87(2007)141–146144accessions using 19RAPD primers yielded 212fragments including 181polymorphic bands (85.38%),on average of 11.16bands per primer (Table 2).In the ISSR generated dendrogram (Fig.1),38accessions are distinctly separated into two major groups,N.lutea (group I)and N.nucifera (group II),at the Jaccard similarity coefficient level of 0.785.The Jaccard similarity coefficient ranges from 0.785to 0.968.In the group II,group IIA includes the hybrids of the two species and group IIB includes the rest of these accessions.In the IIB,it shows four distinct groups at the similarity coefficient level of 0.877,flower lotus accessions cluster in group IIB1,IIB2and IIB3,rhizome lotus accessions cluster in IIB4,and seed lotus accessions (samples 1and 24)are interspersed among the flower lotus.In the flower lotus accessions,wild flower lotus accessions cluster in the group IIB1,Chinese antique lotus (sample 9)and Japanese antique lotus (sample 10)cluster together at 0.915;big-flower type accessions (samples 25,26,27,28,30,31,32,33,35,36,37)cluster in the group IIB2with the exception of sample 31;medium-small type accessions (samples 14,15,16,19,20,21)cluster in the group IIB3with the exception of sample 17.Rhizome lotus accessions (samples 2,3,4,5,7,8)cluster in the IIB4with the exception of sample 6.According to the RAPD data,the dendrogram (Fig.2)indicates that the 38accessions are also distinctly separated into two major groups,N.lutea (group I)and N.nucifera (group II)at 0.654.The Jaccard similarity coefficient between all the accessions ranges from 0.654to 0.956.In the group II,group IIA includes the hybrids of the two species and group IIB includes the rest of these accessions with the exception ofsample 10.In the group IIB,it shows a more-complicated genetic relationship,rhizome lotus accessions cluster together and there are not obvious confines in flower lotus accessions.4.DiscussionIn N.nucifera ,the hybrids of the two species and the rest of these accessions have obvious genetic variation.This is in agreement with the classical morphological identification.Via traditional artificial selection three big groups,viz.flower lotus,seed lotus and rhizome lotus have been formed in N.nucifera .In our study,flower lotus and rhizome lotus have show dissimilar characteristics,which is indicative of their distinct genetic differentiations.Seed lotus accessions do not form a distinct group but are interspersed among the flower lotus.This indicates that seed lotus is close to flower lotus and they might have originated from close wild lotus in genetic relationship.In the flower lotus group,big-flower type accessions and medium-small type accessions display clear genetic variation,indicating that height is an important criterion in the classification system of flower lotus.In our ITS analysis,Chinese and Japanese lotus comprise a single group with a moderate bootstrap support,which indicates there is very high similarity between them.Moreover,according to the ISSR dendrogram,in N.nucifera ,there is no distinct borderline between Chinese and Japanese lotus.Additionally,Chinese antique lotus (N.nucifera cv.‘Chinese Antique Lotus’)and Japanese antique lotus (N.nucifera cv.‘Ohga Lotus’)cluster together.These results indicate thatthereY.-C.Han et al./Aquatic Botany 87(2007)141–146145is very close genetic relationship between Japanese and Chinese lotus.This is an agreement with available historical evidence that the lotus was introduced into Japan by way of Korea from China about500AD.AcknowledgementsWe thank Prof.Qingfeng Wang and Dr.Jinming Chen(of the School of Life Sciences,Wuhan University)for their help with data analysis.We are especially appreciative of the editor-in-chief George Bowes and the reviewers for their helpful comments and detailed suggestions.This work was supported by the Key Technologies R&D Programme of China(No. 2002BA546C).ReferencesBaldwin,B.G.,Sanderson,M.J.,Wojciechowski,M.F.,Campbell,C.S.,Dono-ghue,M.J.,1995.The ITS region of nuclear ribosomal DNA:a valuable source of evidence on angiosperm phylogeny.Ann.Mo.Bot.Gard.82,247–277.Baldwin,B.G.,1993.Molecular phylogenetics of Calycadenia(Compositae) based on ITS sequences of nuclear ribosomal DNA:chromosomal and morphological evolution reexamined.Am.J.Bot.80,222–238.Blair,M.W.,Panaud,O.,Mccouch,S.R.,1999.Inter-simple sequence repeat (ISSR)amplification for analysis of microsatellite motif frequency and fingerprinting in rice(Oryza sativa L.).Theor.Appl.Genet.98,780–792. Borsch,T.,Barthlott,W.,1994.Classification and distribution of the genus Nelumbo Adans.(Nelumbonaceae).Beitraˇge zur Biologie der Pflanzen68, 421–450.Diao,Y.,Liu,J.Y.,Yang,G.X.,Hu,Z.L.,Zhou,M.Q.,Song,Y.C.,2005.Karyotype analysis of the Nelumbo nucifera and Nelumbo lutea by chromo-some banding andfluorescence in situ hybridization.Korean J.Genet.27, 187–194.Divaret,I.,Margale,E.,Thomas,G.,1999.RAPD marlers on seed bulks efficiently assess the genetic diversity of a Brassica oleracea L.collection.Theor.Appl.Genet.98,1029–1035.Doyle,J.J.,Doyle,J.L.,1987.A rapid DNA isolation procedure for small quantities of fresh leaf tissue.Phytochem.Bull.19,11–15.Gilbert,J.E.,Lewis,R.V.,Wilkinson,M.J.,Caligari,P.D.S.,1999.Developing an appropriate strategy to assess genetic variability in plant germplasm collection.Theor.Appl.Genet.98,1125–1131.Han,Y.C.,Teng,C.Z.,Zhong,S.,Zhou,M.Q.,Hu,Z.L.,Song,Y.C.,2007.Genetic variation and clonal diversity in populations of Nelumbo nucifera (Nelumbonaceae)in Central China detected by ISSR markers.Aquat.Bot.86,69–75.Hess,J.,Kadereit,J.W.,Vargas,P.,2000.The colonization history of Olea europea L.in Macaronesia based on internal transcribed spacer1(ITS-1) sequence,randomly amplified polymorphic DNAs(RAPD),and inter-simple sequence repeats(ISSR).Mol.Ecol.9,857–868.Huang,X.Q.,Chen,J.Y.,Huang,G.Z.,1992.Preliminary studies on biosys-tematical relationship between the two Nelumbo species.Acta Hort.Sin.19, 165–169.Kosman,E.,Leonard,K.J.,2005.Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid,diploid, and polyploid species.Mol.Ecol.14,415–424.Les,D.H.,Garvin,D.K.,Wimpee,C.F.,1991.Molecular evolutionary history of ancient aquatic A88,10119–10123. Li,W.G.,Shen,J.J.,Wang,J.B.,2005.Genetic diversity of the annual weed Monochoria vaginalis in southern China detected by random amplified polymorphic DNA and inter-simple sequence repeat analyses.Weed Res.45,424–430.Mattioni,C.,Casasoli,M.,Gonzalez,M.,Ipinza,R.,parison of ISSR and RAPD marks to characterize three Chilean Nothofagus species.Theor.Appl.Genet.104,1064–1070.Moreno,S.,Martin,J.P.,Ortiz,G.,1998.Inter-simple sequence repeat PCR for characterization of closely related grapevine germplasm.Euphytica101, 117–125.Mukherjee,P.K.,Saha,K.,Das,J.,Pal,M.,Saha,B.P.,1997.Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera.Planta Med.63, 367–369.Nebauer,S.G.,Castillo,D.,Agudo,L.,Segura,J.,1999.RAPD variation within and among natural populations of outcrossing willow-leaved foxglove (Digitalis obscura L.).Theor.Appl.Genet.98,985–994.Qian,J.Q.,2002.Cardiovascular pharmacological effects of bisbenzylisoquino-line alkaloid derivatives.Acta Pharmacol.Sin.23,1086–1092. Ritland,C.E.,Ritland,K.,Straus,N.A.,1993.Variation in the ribosomal internal transcribed spacers(ITS1and ITS2)among eight taxa of the Mimiulus guttatus species complex.Mol.Biol.Evol.10,1273–1288. Rohlf,F.J.,2000.NTSYSpc,Numerical Taxonomy and Multivariate Analysis System,er Guide,Exeter Software Publication,Setauket, New York.Sarah,S.K.,Jeffrey,M.O.,1999.Pollen and anther development in Nelumbo (Nelumbonaceae).Am.J.Bot.86,1662–1676.Sinha,S.,Mukherjee,P.K.,Mukherjee,K.,Pal,M.,Mandal,S.C.,Saha,B.P., 2000.Evaluation of antipyretic potential of Nelumbo nucifera stalk extract.Phytother.Res.14,272–274.Soltis,P.S.,Kuzoff,R.K.,1993.ITS sequence variation within and among population of Lomatium grayi and evigatum.Mol.Phulogenet.Evol.2, 166–170.Swofford,D.L.,2001.PAUP:Phylogenetic Analysis Using Parsimony,Version4.0b8.Sinauer Associates,Sunderland,Massachusetts.Thormann,C.E.,Ferreira,M.E.,Camargo,L.E.A.,Tivang,J.G.,Osborn,T.C., parison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species.Theor.Appl.Genet.88,973–980.Wang,Q.C.,Zhang,X.Y.,Hu,C.G.,1997.New system for lotus variety classification.J.Wuhan Bot.Res.15,19–26.Wolfe,A.D.,Xiang,Q.Y.,Kephart,S.R.,1998.Assessing hybridization in natural populations of Penstemon(Scrophulariaceae)using hypervariable intersimple sequence repeat(ISSR)bands.Mol.Ecol.7,1107–1125. Zou,Y.P.,Cai,M.L.,Wang,X.D.,Xu,B.M.,1998.RAPD analysis of germ-plasm in ancient‘‘Taizi lotus’’and modern Chinese lotus.Acta Bot.Sin.40, 163–168.Y.-C.Han et al./Aquatic Botany87(2007)141–146 146。