Use RAPD Analysis to Classify Tea Trees in Yunnan

Agricultural Sciences in ChinaVol.2 No.11 1290 - 1296November 2003Use RAPD Analysis to Classify Tea Trees in Yunnan65 A.SHAO Wan-fang' , PANG Rui-hua2，DUAN Hong-xing'，WANG Ping sheng' , XU Mci?，ZHANG Ya-ping' and LI Jia-hua'('Tea Department，Yunnan Agricultural Universily, Kunming 650201，P. R. China; 8 Biology Department ,Henan Xinyang Normal University, Xinyung 464000. P. R. China; 3 Tea Research Institute ，Yunnan Academy ofAgricultural Sriences, Menghai 666201, P. R. Chima)Abstract: RAPD assessment on genetic variations of 45 tea trees in Yunnan was carried out. Eight pri-mers selected from 40 random primers were used to amplify 45 tea samples, and a total of 95 DNA bands wereamplified, of which 90 (94, 7%) were polymorphism, The average number of DNA bands amplified by eachprimer was 11.5. Based on the results of UPGMA cluster analysis of 95 DNA bands amplified by 8 primers,all the tested materials could be classified into 7 groups including 5 complex groups and 2 simple groups, whichwas basically identical with morphological classification. In addition, there were some speciations in 2 simplegroups.Key words; Yunnan, Tea trees, RAPD, Genetic diversityTea [Camellia sinensis (L.) O. kuntze] isthe RAPD technology is seldom used and it is al-one of the important economic crops, and China ismost focusing on small-leaf varieties (C. sinensis)the first country to sue tea as a beverage, which is or asexual varietieslo-s?. The 45 tea trees, whichnow popular throughout the world. According towere grown in primary tea producing area in Y un-the reports, many wild tea trees were found innan， were studied using RAPD technology. TheYunnan, China. Some researches from botany, bipurpose of this report is to probe into the affinityology, ccology and biochemistry have testified thatrelationships of tea germplams in Y unnan from theYunnan is the original center of tea trees in theDNA level, and make a basis work for further ex-worldl. Investigation of Yunnan tea germplasmsploring and utilizing the plentiful tea germplasm inshows that Yunnan nearly includes all kinds ofYunnan.primitive type, transitional type and cultivated1 Materials and Methodstype tea trees. At present, 4 series, 37 species and3 varieties have been found in the Section Thea (L)1.1 Plant materidsDyer plant in the world, 31 species and 2 varietiesThe materials consisting of 45 tea trees wereof them were distributed in Yunnan, and 24 spe-collected from the Tea Research Institute, Y unnancies and 1 variety were found only in Y unnan,Academy of Agricultural Sciences and the tea gar-which have occupied 80% of the Camellia sinensisden of Kunming 1999 Exposition (Table 1). Theyof the world[2]. Some of them are valuable and rarewere used for DNA extraction.resources, and they have important scientific re-Methodsscarch value and using potentialities, at the same1.2.1 DNA extraction Total DNA was extracted astime, there is an important function in the varietiesdescribed by Cheng et al10.1..reform about tea trees.1. 2.2 DNA detection Electrophoresis dection: WhenHowever, the research method o[ tea tree isDNA was stained with ethidium bromide, thlimited in morphology, anatomy and ecologyla3.f中国煤化工ff fluorescent lightTYHCNMH GSHAO Wan fang(1957-). Professor. Tel: 86-871 5200050 Fax: 86 871 520003, E-mail: wlshao(@ hotmail comUse of RAPD Analysis 1o Classily Tea Trees in Yunnan1291Table 1 Forty-five tea tres as experimental materialsSerial numberNameOriginl placeScicntific naneTypeT-01Xueye100SimaoVariety (selection) .DuanjiebrihaoSmaoC. sinensis wr. assamica DuanjiebaihaoVariety (sclection)T-03YunmeiSimaC. sinersis var. assamrtca YunmeiGood variety in YunnanT-04Yunkxng10MenghaiC. sinensis var. assamica Yunkang10Good variely in ClhinaT05YunshanIZhnotongVariety (esietion)T-06Mengku-dayechaLincangC. sinensis war. assamica Mengku-dayechaGood variety in ChinaT 07Zhaotong taichaZhaotongC. sinensis Zhaorong taichaLocal wnrietyT-08Yuanjiang- nuochaYuxiC. sinens vuar. Pubilimba Yuanjiang *nuochal.ocal varietyT09YunguiC. sinensis var. asamica YunguiT-10ChangycbaihaoC. sinensis var. assanir ChangyebaihaoGood variety in YunranT11AifengC. sinensis war. assamica AilengGood variely in YunnanT 12TaoxingyeSiamoC. sinenris var. asanrica TaoxingyeVariety (selection)T-13C simensis vr. asumtica 99T.14BoshangintaiC. sinensis ver. assamica BoshangjintaiVariety slection)T-15ZaoyuanyeC. sinensis vuar. assamica ZaoyuanyeVaricty (selectiou)T-16BeohongchaYiliangC. sinersis BaohongchaIocal varietyT17MangshuidayeechaBoashanC sinersis wr. assamica MangshuideyechaLacal varietyT-18DhechaWensharC. dishiensis ZhangNew varielyT 19Old tea tree 1Wild tea treeT-20(ld te tree2 .T21CaneliaDebongCamelliuKindred plantT 22Huanglihe wild teaQujingC. tachangensis ZhangT-23Zijuan .New series (seletion)T-24New series (selcction)T-25Nanjian5DaliC. sinensis var. assa macu Nanjian5Variery (selerion)T 26Spcial large leafNo identifyT-27BabucheWenshanC, sinensis ur. assamica BabuchaLocal varietyT28Zhongshan- dayechaDehongC. sinensisuar. assamica Zhongshan dayecha1.ocal varietyT-29DanuochaChuxiongC. utrothea ZhangNew varetyT-30Zhongshan dayeC. sinensis wur. assamica Zhongshan- dayeLocal varielyT-31Manpenlong-dayechaC. sinensis wr. assamrica Manpenlong dayechaL.ocal varietyT-32DoushachaChuxiungC. sinensis DoushachaT-33BaimaochaC. assamira, uur. pulilimbaLoral varielyT-34NabeichaT-35Tuantian- dayechaBnoshanC. sinensis rar. assamica Tuantian dayechaLocal verietyT-36Xinbua-: shongmaochaC. sinensis Xinbue-shongmaocha1.ocal varielyT-37DatuanyechnT-38Xiaogude dayechaC. sinensis ur. asamica Xiaogude dayechaMengkurdeyeC. sinenris var. assamica MengkurdayeT-40MaandachaC. gymrogynoidesWild tea trceT41Ganlongtan-dayechaC sinensis var. assamica Ganlongtan dayechaT42MadenchaC. sinensis vwur. assamica MadcnchaT.43Hule maojianchaC. sinonsis Hule maojienchaLocal varictyT44Jinping- dayechaHongheC. sinenusis vur. as5amica Jinping-dayechaT-45T ujan-baiyachaC yankiangchaas it was inspired by the UV-light. The fluores-DNA that was diluted 20 times and measure 0D atcence strength and the total dcal of DNA were di-260 and 280 nm with the UV-light photometerrect proportion， comparing the fluorescence(Hitachi UV3000). The ratio of natural doublestrength of the experimental materials with the flu-DNA at 260 and 280 nm should be 1.8 or so. Ac-orescence strength ol the standard sample; thcordir中国煤化工herat 1.8or so，IDNA concentration can be measured.the IfYHCNMHGeDNAcontinsUV-light photometer detection: take 15 μla high" aDsorption peak at Z0U nm. the DNA con-1292SHAO Wan-fang pt alcentration can be calculated according to the for-(unweighted pair group method with arithmetic a-mula: DNA (μg pμl') =(ODxs0 X dilution timesXverages) cluster analysis using Multivariate Statis-50)/1 000. After detection， the DNA samplestical package. The calculate formula is as follows:were stored at - 20C for RAPD analysis.d, =[2(x.- Xx)]421.3 RAPD Amplification1.3.1 Reaction system The reaction were per2 Resultsformed in a volume of 25 μl containing 0. 2 mmol2.1 DNA extraction and detection1.1 of each dATP, dCTP, dGTP and dTTP ob-The OD20/OD2so ratio of samples DNA wastained from Pharmacia Biotech company, 1/10 of10 X buffer, 1.5 mmol L' MgCl2, 1.0 U of Taq. I.62-1.71, which means that the purity of DNAwas higher. The density was about 200 ng μI~' bypolymerase.0. 2 pmol I.' primer obtained fromcalculating. Fig. 1 showed that the total DNA byOPERON Company and 20 - 25 ng of genomicagarose gel electrophoresis was clarity. ComparingDNA. The reaction mixture was overlaid with 25 μlwith ADNA, the DNA molecular weight was big-mineral oil. In order to avoid pollution, contrastger and no RNA, which was suitable for RAPD re-amplification was set in every primer using ddH2Oas template. Amplifications were performed in theaction.Perkin-Elmer 9600 PCR amplification instrumentand each primer was repeated for 2 or 3 times.1.3.2Amplification procedure DNA amplifica-tions were performed in a PCR amplification in-strument with a preliminary program: 94C for 3min denaturation, 94C for 1 min, 38C for 1. 5min, 72C for 2 min extension, 45 cycles; 72C for5 min extension, then stored at 4C.1.3.3Amplification DNA fragments detectionFig. 1 Total DNA by agarose gel electrophoresisAmplified DNA products were separated by elec .trophoresis in a 1.5% agarose gel. Gels were2.2 Selection of the primersstained with 0. 5 mg I.1 of ethidium bromide and40 primers (S41-S60，S361-S380) were usedrunat 60-90 V for 3 h. Fragment patterns wereto amplify 3 materials that were selected randomlyviewed under ultra-violet light at 302 nm and pho-from 45 samples. The results show that among 40tographed for further analyses.primers, 3 primers (S42, S57, S59) have no bands1.4 Statistical analysisand one primer (S365) has a few amplification1.4. 1 Amplification fragments statistics DL2000fragments, which could not be distinguished. 12was used as standard marker to define the positionprimers have shown high polymorphism in the 3of fragments in the gels. Every fragment was onematerials. Therefore, the 12 primers were used tomolecular marker. The amplflied fragments wereamplify all materials and 8 primers ( Table 2)，scored from the higher molecular-weight productswhich produced clear and reproducible fragments,to the lower molecular weight products. Amplifiedwere selected for further analysis.ONA fragments were scored for the presence2.3 RAPD markers identification“1”[9.2 or absence“0”and were made an ExcelThe 8 primers selected from 40 random pri-document for analysis.mers were used to examine the level of deteclable1.4.2 Data disposing The amplified DNA frag-中国煤化工ples The resultments were scored by the presence “1" or absenceg.2) and the total“0”. The dendrogram was constructed by UPGMA anYHCNMH Gailsamples. TheUse of RAPD Analysis to Classify Tea Trees in Yunan1293Table 2 Name, sequence of primers and results of RAPD amplificationName of thePrime sequenceNumaber of .Number ofFrequeney ofprimesamplified bandspolymorpbic bandspolytnorphic bandsS41ACCGCGAAGG100. 0S45TGAGCGGACA1492.9S50GGTCTACACC11:S32CACCGTATLC85.7S362GTCTCOGCAA1292.3S368CCAGCTTAGG1(100.0S364CCGCCCAAACl00.0S366 .CACCTTTCCC9bands amplified by S41, S363, S364 and S366 haveinto seven groups including 5 complex groups and 2polymorphism. In all bands, only 5 bands (5. 3%)simple groups. In each clusters, individuals fromwere non-polymorphic. They were distributed inthe same population formed distinct group. .S45，S362, S50 (each one band) and S52 ( twoFig. 3 showed that there were 32 materials inbands), and polymorphism is 94. 7%. It was higherthe complex group I including C. assamica, C.than that in Cathaya argyrophylla (21. 5%)t13]，Linkrge distancePaeonia suf frulicosa subsp (22. 5%) and Spontaneanand paeonia rockii (27. 6%)[4]. Meanwhile, itVAR41vas also higher than that in Kenya tea treesVAR42VAR35(62%)08! and Korea tea trees (84. 5%)[16j. TheVAR32results showed that there was obvious polymor-VAR29phism on DNA level in the tea trees of Y unnan.VAR36The probably reasons were the cross pollination ofVARI9VARI8tea trees for the long times, the original center ofVAR37VAR26tea trees, the long cultivation history and diversi-VAR22VAR31fied eco-environment in Yunnan.VAR20 .VAR6VARI5VARI4VARI3VARIIVAR9VARI0VAR39VAR34VAR30VAR28VARI2VAR44VAR33VAR24VARESVAR27Fig.2 The amplfied patterns of the prlmers on part of ex-VAR7perimental materialsVAR5VARI7VAR8VAR382.4 UPGMA resultsVAR2Based on the 95 DNA bands amplified by the 8VAR45effective 10-mer oligonucleotides primers, a den-VARIdrogram for relationship of 45 tea trees in Y unnan中国煤化工unweigbhedwas constructed using UPGMA (Fig. 3). Accord-MHCNMHGdistancesing to the dendrogram， 45 tea trees were dividedFig.3 DNA molecular dendrogram of45 tea trees1294SHAO Wan-fang et alsinensis, C. yankiangcha, C. gymnogynoides andvariety selected by nature. Now, Yunkang10 wasC. assamica. var. pulilimba. Except for C. gymnocultivated widely in Yunnan. In the early spring ofgynodies, they all belong lo the Serics Sinensis,2000, the cold harm happened again in the south ofwhich was the reason for them to cluster together.Yunnan, and most of the tea varieties were endan-In complex group I, the tea trees cornmnonly havegered in thal time, bul the injury of Yunkang10long cultivation history. or abundant variation typewas slight. It indicates that this variety has cold-and someexcellent characteristics. Therefore,resistant ability. According to the available pedi .these tea trees are valuable materials in breeding.gree, the variety belongs to C. assamica. GenerallyComplex group 1 included tachungensisspeaking, small leaf variety (C. sinensis) has aZhang，special large leaf tea tree (no identify) andgood ability to resist cold, but Y unkang10 (C. as.Datuanyecha (no identify). It was found that thesamica) showed much better resistance than smallleaf of special large leaf tea tree was larger, fragileleaf variety. From the fact that the Y unkang10and thicker, and it should belong to primitiveformed one independent group and it was close totype. According to the Mr. Zhang's classifyingsmall leaf variety (C. sinensis) and far away tosystem and cluster results, special large leaf tealarge leaf variety (C. assamica) in dendrogram, ittree and Datuanyecha may belong to the Seriescould be concluded that this sample was a very disQuinquelocularis Zhang, and both of them havetinctive variety. It is very valuable to carry out aparticular characteristics.thorough research, which purpose is to obtain ex-Complex group II included Dihecha (C. dishittensive utilization value.ensis Zhang), old tea tree 1( no identify) and Xin-Complex group VI included Baohongcha andhua- shongmaocha(C. sinensis). Dibecha is a newDoushacha. Both of them are good varieties of sevariety of Section Thea (L) Dyer, which originalxual population, and belong to small leaf varietyplace is in Guangnan County of Yunnan, and it be-(C. sinensis). Because of the difference of geneticlongs to the small arbor and large leaf type. Oldmaterials, the affinity relationship of small leaf Va-tea tree I obtained from the tea garden of Kunmingriety (C. sinensis) is far away to the large leaf va-1999 Exposition was 800 years old, which originalriety (C. assamica). So, it is impossible for themplace was in Simao county of Y unnan, and its typeto cluster together with C. assamica in dendro-vas small arbor and large leaf. According to thegram. In addition, it was testified that usingcluster results, it shows that both of them comeRAPD to classify tea germplams was credible.from different areas, but they have similar charac-Complex group VIl included three samples:ters.Ganlongtan-dayecha, Madencha and Tuantian-Only one sample- -Danuocha (C. atrothea Zhang)dayecha. According to the pedigree, they all be-was in group IV. Comparing with the available pedi-long to C. assamica. But Fig. 3 shows that they dogree, it belongs to Pentastylae Zhang. Generallynot be clustered with other C. assamnica ( complexspeaking，the Pentastylae is more primitive thangroup I)，so the affinity relation of them is farothers. Therefore, this sample, forming one inde-away with complex group I. The phenomena per-pendent group， is identical with the traditionalhaps indicated that it is not comprehensive toclassily.classify the tea germplasm just by the morphology.Yunkang10 was the only one sample in groupAt the same time, it showed that there were the a-V. It belongs to the good variety of China and itbundant nature resources in tea trees in Y unnan,was selected by a strong cold harm in 1973 in where was the tea original center in the world.Menghai. Xishuangbanna of Y unnan，China. In中国煤化工ould be gestated inthat situation, all varieties of tea trees died except:MHCNMHGJetic diversily of thefor Yunkangl0. So it was regarded as the valubale tea uets Lal De ecieicu cumprehensively from theUse of RAPD Analysis 1o Clasily Tca Trees in Yutnan1295DNA level. The three samples are valuable to becolony of tea germplasm by RAPD.protected as the special tea germplasm for furtherThe rescarch result of 45 tea materials in Yun-research.nan shows that the polymorphism of Yunnan teaThe affinity relation between the dendrogramtrees is 94. 7%，which was higher than that ofand species, and the affinity relation between theKenya tea trees (62%)[15] and the Korea tea treesdendrogram and varieties will be discussed in fu.(84. 5%)[16]. It indicated that there were abundantture.genetic diversity and distinctive characteristics inthese tea trees on DNA level. At the same time，3 Discussionsome molecular biology foundation would be pro-Eight primers (S41， S45， S50， S52， S362,vided to explore and use tea resources, build germ.S363, S364 and S366) selected from 40 primers canplasm gene information bank and cultivate new andproduce clear and reproducible fragments and begood variety.used to RAPD research directly.In conclusion, using RAPD technology 1o re-45 sarnples of tea trees were divided into sevensearch tea germplasm resources should be a trulygroups while the Euclidean distance was five.method to identify what species or variety the teaComplex group I including 32 samples belongs totree belongs to. In this case, it is signilicant toSeries Sinensis according t0 the Zhang's classifyingfind new species in time, and to protect and usesystem. New variety Dehecha ( C. dishiensisthese germplasm purposefully. Meanwhile， be-Zhang) and old tea tree 1 (no identify) were clus-cause RAPD can produce a number of moleculartered in a group, indicating that they have particu-markers, it is easy to distinguish the varieties ，lar genetic materials compared with C. assamica. .which are very similar in morphology and affinityIn addition, it was very interesting that Danuocharelationship is close by polymorphic. The gene(C. arothea Zhang) and Yunkang10 (C. assamica)bank of tea germplasm can be built with DNAformed one simple group respectively. It ilustrar-polymorphic examination, and it will be helpful toted that the two samples had some distinctive cha-improve and complete the phylogenic researchracteristics from some profile. Especially to theabout Section Thea (L) Dyer. Plant. At one time,Yunkangl0, as the good variety of the country, itsome molecular foundation can be provided for teahas the obvious cold resistance. Therefore, thisbreeding, predominant population building and thesample is undoubtedly valuable for further researchsuperiority model establishing of good variety.and some reference foundation from DNA level canMeanwhile, it was also beneficial to resolve somebe offered for selecting good variety. Two smalldissident problems in the taxonomical study.leaf varieties ( C. sinensis) ( Baohongcha andDoushacha) were clustered together in one groupAcknowledgementsand the affinity relationship of them is far awayThe authors are grateful to the financial sup-from C. assamica. This fact perhaps explained thatport provided by Yunnan Science and Technologyit was creditable to classify the tea germplasm byBureau, and some samples provided by Tea Re-using RAPD. According to the pedigree, Ganlong-search Institute, Y unnan Academy of Agriculturaltan- dayecha, Madencha and T uantian-dayecha be-Sciences, China.long to C. assamica. 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