ofSystematics and Evolution Journal 49(3):169-175(201 1) doi:10.1111/j.1759—6831.2011.00128.X Research Article High universality of matK primers for barcoding gymnosperms 1,2yan LI l,一,4Ram C.POUDEL ILian.Ming GA04 l,3De.Zhu Li 5Alan FORREST 1(KeyLaboratoryofBiodiversityandBiogeography,KunmingInstituteofBotany,ChineseAcademyofSciences,Kunming 650204,China) 2(Institute ofAlpineEconomicPlants,YunnanAcademyofAgriculturalSciences,Lijians 674100,China) 3(GermplasmBankoffieldSpeciesinSouthwestChina,KunmingInstitute ofBotany,ChineseAcademyofSciences,Kunmjn9650204,China) 4(The Graduate University ofChineseAcademy ofSciences,Beijing 100049,China) 5(Royal Botanic Garden Edinburgh.Edinburgh EH3 5LIL UK) DNA barcoding is a tool to provide rapid and accurate taxonomic identification using a standard DNA two-marker combination of motK+rbcL was formally proposed as the core barcodc for land plants by region.A the Consortium for the Barcode of Life Plant Working Group.HoweveL廿1ere are currently no barcoding primers Abstract for matK showing high universality in gymnosperms.W-e used 57 gymnosperm species representing 40 genera.1l families and four subclasses to evaluate the universality of nine candidate matK primers and one rbcL primer in this as a universal primer for gymnosperms due to high universality. One ofthe nine candidate matK primers(Gym_F1A/Gym_R1A)is proposed as the best‘、miversal”matK primer for gyrrmosperms because ofhigh polymerase chain reaction success and routine generation ofhigh quality bidirectional well on the sequences.A specific mark primer for Ephedra was newly designed in this study,which study.Primer f l F/724R)of而吐is proposed here performed sampled species.111e primers proposed here for rbcL and matK be easily and successfully can amplified for most gymnosperms. Key words DNA barcoding。matK,gymnosperm,primer universality. DNA barcoding is technique to provide rapid and a specific DNA a accurate taxonomic identifcation using region(Hebert&Gregory,2005).it has become a useful tool for species identification(Kress&Erickson.2007; Erickson et a1.,2008),and discovering new or cryptic species(Newmaster&Ragupathy,2009;VralentiIli et a1.. 2009).The mitochondrial cytochrome c oxidase subunit 1(coJ『1 has been used as titleation in many animal a barcode for species iden— groups(Hebert et a1..2003). ever,universality of low in some matK primers was reported to be studies(Sass et limit its use et a noristic study was reported of matK primers for a1.,2009).Improvement increase universality in angiosperms,and recommendation as part of the barcode(Lahaye et a1.,2008;CBOL Plant Work- barcoding can was reason a core key for its Group.2009).Although high ing suitable for barcoding to the search substitutions in matK make it for alternative et a1.,2005; Cowan et a1.。2006;Fazekas et a1.,2008;Hollingsworth et al。2009).Recently,a two—marker combination of matK+rbcL was recommended as the core barcode for C:onsortium land plants bv the (CBOL)(CBOL Plant for the Barcode of Lifc Working Group,2009). Universality ofprimers for polymerase chain Feac— tion(PCRl and sequencing is one ofthe most important criteria for DNA a1.,2009),which may barcodc.For example.69%success for matK in recovery (Kress as a However,the low substitution rate of CDl makes it tin- plants.and has led barcoding regions(Kress a1.,2007;Fazekas et a1., 2008;Fbrd et a1.。2009;Kress et a levels of nucleotide good barcode marker in terms of species identification.it is diffcult to routinely amplify and sequence across divergent lineages and as such more studies to develop‘‘universal’’matK primers are necessary(Kress&Efickson.2007;Hollingsworth, Plant Working Group。2009;Ford et a1., 2008;CBOL 2009).Recently,order-specific matK primers for an- giosperms were suggested by Dunning&Savolainen (20 l O).Nevertheless.there are still significant chal. a1..2007;Kress 1enges that need to be overcome for high—throughput &Erickson.2007;Ford et a1.。2009;Hollingsworth et a1..2009).In the core barcode,the primers for rbcL floristic studies using this DNA region for barcoding show a barcoding(Chase et Gymnospenns are a group of seed-bearing plants mat consist of four subclasses,Cycadidae,Ginkgoidae, Accepted:23 February 201 1 correspondenee.E.mail:gaolrn国mail.kib.ac.ca;Tel.:86-871- Gnetidae.and Pinidae,representing 8 orders,1 2 families,83 genera,and approximately 990 species (Christenhusz et a1..20 1 1).Gymnosperms are all woody Plant W-orking Group.2009;Kress et Received:23 December 2010 +Author for plants. plants(CBOL a1.,2009).How- high level of universality in land 5223505;Fax:86・871-5217791. @201 1 Institute ofBotany,Chinese Academy ofSciences 万方数据 trees。shrubs or lianas.They grow throughout most ofthe 170 Journal ofSystematics and Evolution world,and V01.49 No. the dominant vegetation in many colder are and arctic regions(Judd et a1.,2007).Gymnosperms have major economic uses:many are important wood resources and omamental plants.In China.there are 3 201 1 of Botany,Chinese Academy of Sciences(KUN).Leaf material was collected in the feld or from Kunming Botanic Garden.and immediately dried in silica gel til laboratory analysis. un- about 250 gymnosperm species in 34 genera and l 0 families(Wu&Raven.1 999).For gymnosperms.there is no universal specific matK primer for DNA barcoding that has performed well to date.Several matK primers 1.2 Candidate primers screening strategy The nine candidate matK primer pairs and proposed for barcoding land plants show low levels of PCR success in gymnosperms fCBOL Plant W6rking nine matK primer pairs Group。2009;Ford et a1.。2009;Liu et a1.201 11.For matK primer pairs example.the PCR success rate is less than 50%in gym— nosperms based on data compiled from severallabora- toties(CBOL Plant working Group,2009),and only 33%PCR success tor pnmer pair et was obtained with the best matK sampled a1.r2009).The gymnosperm fact that have been developed as no species by pord specific matK primers barcodes for gymnosperms may not have are those most commonly used for species representing the 11 families for PCR amplifi— Cupressaceae.two samples from each were included in this family—level assessment(see success amplification is essential for its application and cation with aU candidate matK primers and one rbcL primer as contr01.For the large families Pinaceae and velopment to improve the overall of matK recently designed barcoding land plants.We adopted two steps to test the universality of candidate primers.First。we selected 14 candidate rate are been tested systematically,and the remaining three explain the low amplification of this region.Primer de. success sin— a gle而cL primer pair are outlined in Table 2.Six of the 1’able l 1.Second matK primers showing more than 50%PCR were used for further assessment all remain. on ing ofthe nine matK primer pairs met this universal plant samples(five requirement). matK primer for the whole plant kingdom because of 1.3 DNA extraction.amplification,and sequencing Genomic DNA was extracted from silica.dried leaf as part ofthe barcode(CBOL Plant Working Group. 2009).However,it is a challenge to develop a universal an extensive variation within the DNA region(Kress &Erickson 2007).Further primer development efforts are required in non—angiosperms for maⅨfCBOL Plant material using the CTAB method Wbrking Group.2009). buffer r 1 0 mmol/L Tris.HCI。pH 8.0,1 mm01/L EDTA) to a final concentration of 40一50 ne;/“L to avoid any In this study,we used nine candidate matK primers and one而cL primer to evaluate the PCR and sequencing vailation in PCR universality for gymnosperms.Six ofthe nine differences. matK primers candidate recently designed with five as yet an— the remaining three were proposed as “universal”primers for barcoding land plants.W色se. 1ected 57 species.representing 40 genera of 1l families tllat correspond to all four subclasses of for trial in this study.Our aim here is to universality of the matK primers for gymnosperm evaluate the gymnosperms. described by Doyle due to success DNA concentration A11 PCR reactions were carried out are published,and as &Doyle f1 987).The genomic DNA was dissolved in TE PCR System 9700 ter thermal City,CA,USA)in contained 10 tzL 2 a on a cycler(Perkin GeneAmp Elmer,Fos. total volume of 20“L,which Taq PCR Master Mix f0.1 U Taq x polymerase//zL.0.5 mmol/L each dNTE 20 mmol/L Tris—HCl(pH 8.3),1 00 mmoI/L KCl,3 mmoI/L MgCl2: Tiangen Biotech,Beijing,China),0.2/zL bovine serum albumin(1 0肛g/uL),0.5/zL each of the forward and reverse primers(1 0/zmoI/L),and l“L template DNA. Material and methods Negative controls were run alongside all PCR reactions. The matK thermocycling profile was:94。C for 3 min.35 1.1 Sampling strategy A total of 57 species representing 40 genera.11 families,and 8 orders were selected in this study(Ta- cycles of94。C for 30 s,52。C for 30 s,72。C for 45 s,with ble 1),covering approximately 47%of genera,92%of 50 s.52。C for 1 min,72。C for 1 min,with families。88%of orders。and 1 00%of the subclasses sion of72。C for l 0 min.The PCR products were visual. ized on 1%Tris-acetate_EDl'A agarose gel stained with of era gymnosperms.These and families samples represent all the gen. in China.To represent the OCCu币ng a final extension of72。C for 1 0 min.The rbcL thermocy. cling profile was:94。C for l min.30 cycles of94。C for ethidium bromide alongside a a final exten. GeneRuler 1 00 bp DNA wider diversity of larger genera rthose containing>20 ladder(rermentas。Glen Burnie,MD’USA).The species occurring in China),more than one species per genus were included in the analysis.Voucher specimens products were purified using ExoSAP.IT fGE Health. were deposited in the herbarium of Kunmmg Institute carried out in PCR care,Cleveland,OH,USA).Sequencing reactions were a total volume of 6“L containing 0.2“L @201 1 Institute of Botany,Chinese Academy of Sciences 万方数据 LI et a1.:marK primers for gymnosperms 171 Gym_F1A Gym_F2A Gym_FIB Gym_F2B P刚 Gym_R1A Gym_R2A Gym_RIB Gym_R2B PKRl Glm-06032 ++ ++ ++ + + Cycadidae Cycadaceae Cycadidae Cycadaceae Cycas guizhouens括t Cycas micholitzii Ly・012 ++ ++ ++ + ++ Cycadidae Zamiaceae Encephalartos lehmann舻Ly-014 ++ ++ ++ + + Ginkgoidae Ginkgoaceae Ginkgo 一 + + + + Gnetidae Ephedraceae Gnetidae Ephedraceae Ephedra gerardiana Ephedra intermedia Gnetidae Gnetidae Ephedraceae Gnetaceae Pinidae Araucariaceae Gnetum pendulumt Araucarla bidwillii Pinidae Araucariaceae Araucaria cunninghamiP Pinidae Araucariaceae Araucaria heterophylla Pinidae Cupressaceae Pinidae bff0幻’ Ephedra likiangensis.I Ly一028 090937—‘—。———。—。 Sunh-zx一1688 一 一 一 一 一 Ly-024———。—。—。—。 ++ ++ GrBOWS0801 ++ 一 一 Ly—Ol 5 ++ 一 ++ + ++ Ly一016 ++ 一 ++ + ++ ++ 一 ++ + ++ Calocedrus macrolepis Ly—004 Glm—103074 ++ 一 ++ + 一 Cupressaceae Chamaecyparis加rmosensis Glm-103065 ++ ++ ++ 一 + Pinidae Cupressaceae Ly-007 ++ ++ ++ + ++ Pinidae Cupressaceae Cunni,29hamia lanceolata Cupressus duclouxiana 一 ++ + + Cupressaceae Cryptomeriajaponica Ly一01l Glm一103142 ++ Pinidae ++ 一 十+ + ++ Pinidae Cupressaceae Folaenia hodginsii Ly-006 ++ ++ ++ ++ ++ Pinidae Cupressaceae Ly-005 ++ ++ ++ ++ ++ Pinidae Cupressaceae Glyptostrobuspensilis dun由eru¥pingii vaL wiIsonii 一 ++ 一 一 Cupressaceae ++ 一 十+ 一 一 Pinidae Cupressaceae dunit,erus squamatat Metasequoia glyptostroboidest Ly一026 G1m-082126 ++ Pinidae Ly—002 ++ ++ ++ + ++ Pinidae Cupressaceae Platyctadus orientalis Ly一020 ++ 一 ++ + 一 Pinidae Cupressaceae Ly一021 Glm-l 03046 ++ ++ ++ + + ++ ++ ++ + ++ Glm-09241 1 ++ ++ ++ ++ ++ Ly一009 ++ + ++ + ++ Ly・019 孤“缸suwhuenens括Ly-01 8 ++ ++ ++ + ++ ++ ++ ++ ++ ++ Glm-102904 ++ ++ ++ ++ ++ Ly-017 ++ + ++ ++ ++ Ly_0口9 ++ ++ ++ ++ ++ Pinidae Cupressaceae Sequoia sempervirens Taiwania cryptomerioides Pinidae Cupressaceae Taiwania砌usiana Pinidae Cupressaceae Taxo硪um distichum Pinidae Cupressaceae Thujopsis dolabrata Pinidae Pinidae Cupressaceae Pinaceae Pinidae Pinaceae Pinidae Pinaceae Cathaya argyrophylla Cedrus deodara Pinidae Pinaceae Keteleeria davidiana Ly-001 ++ + ++ + ++ Pinidae Pinaceae Kete彪eria evelyniana Ly一003 ++ + ++ ++ ++ Pinidae Pinaceae Keteleeriafortunei ++ + ++ + 十十 Pinidae Pinaceae ++ 一 十+ ++ ++ Pinidae Pinaceae Lar/xpotaninii var.chinensis Larixhimalaica Ly・010 G1m-06081 Glm-081604 ++ 一 ++ + ++ Pinidae Pinaceae Pinidae Abies george: Glm一08912 ++ 一 ++ + ++ Pinaceae Picea liMangens括 Picea smithiana GIm-08 1 533 ++ 一 十+ + ++ Pinidae Pinaceae Picea spinulosa Glm-08 l 883 ++ 一 ++ ++ ++ Pinidae Pinaceae Pitills arman硪f Ly・027 ++ 一 ++ ++ ++ Pinidae Pinaceae Pinus wa肼chiana 81471 ++ 一 ++ + ++ Pinidae Pinaceae Pinus yunnanens捃t Ly一025 ++ + ++ ++ ++ Pinidae Pinaceae Pseudolarix amabilis ++ + ++ ++ ++ Pinidae Pinaceae ++ 一 ++ 一 十 Pinidae Pinaceae Pseudotsuga sinensis Tsuga dumosa Ly一008 Glm—l 03063 Glm一081797 ++ 一 ++ 一 ++ Pinidae P0docarpaeeae Dacrycarpus imbricatust Ly一023 ++ ++ ++ ++ + Pinidae Podocarpaceae Nageiafleuryi ++ ++ ++ ++ ++ Pinidae Podocarpaceae Nageia nagi Ly一022 Ly-01 3 ++ ++ ++ ++ 一 Pinidae Podocarpaceae Podocarpus macrophyllus GBOWS0294 ++ ++ ++ ++ 一 Pinidae Podocarpaceae Podocarpus Glm062 11 ++ ++ ++ + 一 Pinidae SciadopitVS verticiff4衄t Amentotaxus argotaenia G1m-09224l ++ + ++ + + Pinidae Sciadopitvac七ae Taxaceae Pinidae Taxaceae Cephalotaxus Pinidae Pinidae‰【aceae Taxaceae Cephaiotaxus wUsonianat Pseudotaxus chienii Pinidae Taxus wfifllichianat mannii 1 82 ++ ++ ++ ++ 十 ZSD001 ++ +十 ++ ++ ++ Glm-103119 ++ ++ ++ ++ ++ Glm-07549 ++ +十 ++ ++ ++ RC l 289 ++ ++ ++ + ++ ++ ++ ++ ++ ++ Torreya力rgesll var.yunnanensis GLM一092567—18 evaluatir PCR success using SUCCESS "to to evaluate e and coverage and em in this this study.伶pccles selected tor selected for ties s inn bold were e selected for i sec c p S study._|-Species ni s evaluating I"UK u sequencing sequence quality Pinidae St Taxaceae neriifolius Taxaceae the nine candidate matK primer sets at family-level.一,no band;+,weak band;++,strong band. @201 1 Institute ofBotany,Chinese Academy ofSciences 万方数据 172 Journal of Systematics and Evolution V01.49 No. 3 201 1 Polymerase chain reaction primers used for screening universal primers for gymnosperms in this study Table 2 Gene Primer name rflatK Gym—R1A Gym FlA Direction f 5’.TCA YCC GGA RAT TTT GGT TCG.3 r 5’一ATYGYRCTTTTATGTTTACARGC.3’ Gym—R2A f 57-ACYTTTCGYYRCTGGATCCAAG.3’ This study,designed by Alan Forrest This study,designed by Alan Forrest This study,designed bv Alan Forrest GvIll F2A r 5’一GTTTTAGCRC—汀GRRAGTCGAAG.3’ This Gym—RlB f 57一TCA TCC RGA AAT TTT GGT KCG.3’ r 5’.ATMGTACTTTTATGTTTACARGC.3’ f 57-AyYTTTCGTCGCTGGATCCGAG.3 GⅧ_F2B r 5’.GTT 11A GCR CAT NY552F f 5’一CTGGATYCAAGATGCTCCTT-3’ NYll50R r 5’.GGT CTT TGA GAA GAA CGG AGA.37 Damon Linle,unpublished Damon Little,unpublished PKF4 f 5’-CCCTATTCTATTCAYCCNGA.3 Fazekas et PKRl r 57-CGTATCGTGCTTTTRTGY丁丁.37 Fazekas et 5R f 57-G11 CTA GCA CAA GAA AGT CG一3 XF Gym_FlB Gvll-1 R2B study,designed by Alan Forrest This study,designed by Alan Forrest This study,designed by Alan Forrest This study,designed by Alan Forrest GRW AIrl’RRA AG.37 This 7 study,designed by Alan Fotiest Ford et Ford et al r20081 al f2008) a1.(2009) a1.(2009) r 57.TAA TTT ACG ATC AAT TCA TTC.3’ 3F_KIM 1R KIM f 57一CGT ACA GTA CTT TTG TGT TTA CGA G.3 r 5'-ACC CAG TCC ATC TGG 1326R f 5'-TCT AGC ACA CGA AAG TCG AAG T-3’ Ki-Joong Kim,unpublished Cu6noud et a1.2002 r 5'-CGA TCT ATT CAT TCA A1-A TTT C.37 Cu6noud f 5'-ATG TCA CCA CAA ACA GAA AC.3’ Fay r 5'-TCG CAT GAT CCT GCA GTA GC.3’ Fay et a1.1997 390F rbcL Source Sequence(5'-3 7) lF 724R AAA 7 Ki—Joong Kim,unpublished TCT TGG TTC.3’ et et a1.2002 a1.1997 e forward;r’reverse.Damon Little is based at New York Botanic Garden,New№rk.Ki—Joong Kiln is based at Korea University,Seoul purified PCR product,0.1 5雎L BigDye terminator sequencing pied 1 4 species at family level(Table 1 1.The universal. ity for the nine matK primers showed a much more and variable PCR mixture(V3.1),1.2雎L sequence buffer, 1.4“L“mol几primer.The cycle sequencing pro— file was 32 cycles of96。C for 10 s.50。C for 5 s.and 60。C for 4 min.The purified sequencing products were run on an ABI 3730xl automated sequencer(Applied RlA。Gym 1.4 Sequence quality and coverage analysis Sequences were trimmed using a window size of 20 bp’with segments with 2 bp showing<20 fQV an established metric for Qual. determining quality sequencing data)trimmed.Sequence quality was assessed using Sequencing Analysis 5.3.1 software (Applied Biosystems).The sequence quality recovered was defined such that both the forward and reverse lengths should be>50%ofthe original read length and the assembled contigs should have>50%overlap in the alignment of the forward and reverse reads with<1% low—quality bases(<20 QV)and<1%internal gaps and substitutions when aligning the forward and reverse reads.The percentage ofbases with>20 QV>30 QV and>40 OV was determined using Sequencher 4.9 Codes.Ann ities for l 。 Arbor,MI,USA).Error PCR universality at genus R l, probabil. sequences were calculated after base pair calling using Phred as implemented in Aligner 5.3.6 consensus level(50.0%一92.9%、and family level(75%一1 00%),respectively.The remaining four matK primers(3F KIM/lRⅪM,390F/1326R, XF/5R.and NY552F仆TY“50R)showed low PCR SUC. cess(0.0%一1 4.3%1.These four primers with low PCR success were rejected for further evaluation on the ex— panded sample set. On all 57 sampled reads should have high length of read:after trimming to an average Qv>20 in a 20.bp window,the post.trim (Gene 14 ..F2A/Gym..R2AKFG4/PymKRFl)B/Gym P myG/B2showed Band 2R. F_myGhigh B Biosystems,F(Ister City'CA,USA). ity Value rate(0.0%一92.9%)on the success selected species in the family level assessment.Five of the nine candidate matK primers(Gym F.1 A/Gym 1 00%PCR with species.而吐performed universality(Table well 1 1.For the five candidate matK primers used for further evaluation. Gym_F1A/Gym_R1A showed the highest PCR success with 94.7%.followed by Gym FlB/Gym RlB with 93.0%。Gym F2B/Gym R2B with 80.7%.KF4/PKRl.P with 77.2%.and Gym F2A/Gym R2A with 57.9(Ta— ble l 1.However,three primers Gym F2A/Gym I也A. Gym P BKf 4/P B2and R_myG/ 2F_。 lRK . yielded weak PCR bands of25.7%,53.1%,and 21.3%.respectively, for the 57 species.A11 the sampled species of sampled family Ephedraceae failed to amplify using these five matK primers(1'able 1). (CodonCode,Dedham,MA,USA). 2 Results 2.1 Primer universality The universality ofprimer for rbcL(1 F/724R)per- formed well with 1 00%PCR SUCCESS on a11 the sam. 2.2 Sequence quality and coverage To assess the quality of generated traces sequence for matK primers Gym_F 1 A/Gym_R 1 A and Gym_F 1 B/Gym_R 1 B,which success and showed PCR high PCR selected nine bands,we s仃ong species @201 1 Institute ofBotany,Chinese Academy ofSciences 万方数据 LIH al:mark pfimcBfor∞mno举m§I 删b嘲■ RI㈣描■ G”.R1B㈣■ 73 ■P●@1,m7blw ■oⅢ』1^1≈佃1粥 ■o"Ff81≈1∞1Ⅵ 却n 互Z互Z翌五Z五Z互Z■匝匠五Z互Z2五Z乙Z互Z蛋五蛋乙Z疆哐互互翌互l Ⅻ.F∞㈣”■ 鲫.F凯●∞b511■ 523 ■Ⅻm'Ⅻb1,¥16 -0m F执㈨tj” 。R。R-。L。黑盎篇篇詈嚣:篇嚣嚣墨饭:蹴篇篇蒜:咣镒嚣端嚣盘哥矗鼢譬嚣 PF,Y4q*I诅I.K¥p州1wM for sequencing,representingthe nine sampledfamilies except for Ephedraccae,Gnemceac.and Ginkgoaceae due to PCR failure A¨the generated 36 bidirectional coverability,sequence quality,andlevel ofspaciesdis- criminationfCBOL PlantⅦbrkingGroup,2009)Hl曲 sequencetraces shoⅥed high sequence quality.withfew PCR amplification success of rb吐for gyrrmosparms has been reportedln some studies rCBOL PlantWork— Iow-quality bases trimined oul The amplicon sizes of ing Group,2009,Liu the primer pairs were 876 bp including primer sites Length of read for sequence u'dces generated by primer Oym_FIA/Gym—R】A ranged from 799 to low in gymnosperms versalily Inthis study,PCR universality ofrbcLhereperformed best with 100%PCR success thr all the 57 sampled 829 bp with high QV f120J.and 779 8i6 bp with mghQV(120Jforthe sequences generated by primer Gym F1B,Oym—R【B Thcre wcrc 93 7%97 2%and species representingl】of】2fmmiliesofgymnosparms SoⅥproposcdprimerlF/724R asthe“universal”rbcL DA'O 82 8%-92 5%ofbases(OV)30).and 9l 3%一96 2% and 83 7即904%of bases(Qv)401向r primer pairsGym—FIA/Gym—RlAandGym FIB/Gym_RlR, respectively.On the whole the sequence quality of Gym F1~日ym—RlA a】itflc ls better than that of Gym FiB,Oym—RlB The assembled contigs sfioⅥed >70%overlapmthe alignment oftheforward and re— verse readswitfioutIow<lualiⅣbases amonggenerated Position and orientationofmark primen Position and orientation of the m-e candi. al,2011).wfiereas PCR uni・ primer forbareoding gymnosperms The hi曲ly vail曲ie,删坼region has Iower PCR amplification SUCCESS than the mote conSUl%.ed rbcL genc fKress&Erick,^m Ing Group,2009)It D“fy m口tK with a 1s 2007 CBOL PIam Work- currently not possible to am- single universal primer pair across the whole plant kingdom to ncilitate high throughout、 rapid,automated,and cost-e舵ctive species ldentifiea・ tion fKress&Erickson 2007:Dunning&Savolainen. 20lOl Three,M,K primer secs namely 390F/l 326R (Cednoud 2.3 et ofmarKⅫrelatively et aI.2【)02I XF‘5R fFord c【al 2009)and ”一KIⅥlR—KIM(K—J Kim.KoreaUniversi眦Seoul. unpubl data,also Sec Hollingsworth 2008)werc pro・ fo㈨nfK with(Fnhnl{}|…k?n posed as currently the best“universal”脚tK primers mr land plant fhgp://www.barcoding si ed“PDⅣ template ls Illustrated m Fig l Primer Gym Fl~bym RIA and Gym—FlB/G),xn RIB Io— calcd at the same position with a length of832 gp be. twcen primer sets Primers Gym F2A/Gym—R2A and performed well for most groups of angiosperm they shcm'edlow PCR SUCCESSfor sampled species ofgym- date primers lune,as Gym F2B’|G)m—R2B also share an Idenfenl position wIth a length of 782 bp between primer se【s(Fig l h Forward primerPKF4 S Latted alpositloff 537 bp andthe w㈣primerPKR4 at 1 bctwccn primer scts(Fig 327bpwith alel39th of770 bp al 0 as the ptant colt:barcode based on its 201IInstilBte ofBotan*Chme"Academy ofSciences 万方数据 three,w,K unsuitabfe as“universal”mdtiC primersfor gymrlosperms Two primers ofthe nine eandidale mdfK primers. and Gym_FIB/Gym—RIB, Gym—FIA/Gym—RlA produczs岛r sampled species Ofthetwomd,K primer pairs,primer Gym F】丸'Gym—RlA,performed bene^ successfully amplifyingallthcll sampledfamifies and Primer uni、ersalgy is the mosl important crite- for DNA baK(mmg in the^nl instance(Chase ommcnded primers are shoued highIevels 2007;Kress&Erickson 2007;Ford et al 2009; fIo[Iingsworth et al 2009)rbcL+matK has bccn mc- el nospcrm m this study.1ndicaring that these of时R universality.high sequence and ty qua coverage.and could a[so yield swong PCR 11 3 Discussion rion Informaoononbarcodeloci pdn Althou曲theseprimers re・ 39 oul ofthe 40 sampledgeneraf97 5%1 or如hedra(Gnelales:EphedmceacI Only species ampli竹 fTabIe”For primer pair G)wa—FIB,Gvm_RIB.37 falIed to of 40 genera f92 5%l”m ampllfled successfully. but species of two sampled families fEphedraeeae 174 ofSystematics and Evolution Journal and Ginkgoaceae)could 1).Based not study(Table be No. V01.49 in amplified this assessments of primer on universality and sequence quality and coverage,we propose that primer Gym_F 1 A/Gym_R1 A currently the represents best universal primer for matK in gymnosperms- of an appropriately short sequence to facilitate DNA extraction and length(300-800 bp) amplification(Kress and orienmtion of the five candi. al。2005).Position date marK primers showing high PCR success is given in Fig.1.The length ofthe proposed matK barcode region for primer Gym_F 1 A/Gym_R 1 A is 832 bp long using fortunei matK sequence(AF228 1 09) the Cephalotaxus as reference(excluding primer sequence).The a size of mis product is amenable to the acquisition of bidi. rectional sequence reads with a single primer pair,thus meeting the criterion of DNA barcode length. As none of the candidate matK primers amplified samples ofEphedra,a specific matK primer for Ephedra (Eph F: 57一TCATTCAGAGCTGTTAGTTAG.3 5'- .h17pCEGA.TCGAT CATG A:R 7. 3 newly designed in this study.This primer per- formed very well on the three sampled species of was Ephedra with located at a Gym l 00%PCR success.This similar position the matK gene on primer is as primer A/Gym. A. the ength of the IF R1, 1 ensuring that gymnosperms barcode region for to align.The primers is similar and easy proposed in this study for barcode rbcL and marK can core be easily and successfully amplified for all the genera of gymnosperms in China. We hope our work will facilitate DNA barcoding of gymnosperms worldwide as Wre well. to Dr.Chun.Xia Acknowledgements grateful ZENG.Mr.Jun.Bo YANG。Mrs Jing YANG,and other are colleagues for laboratory help also thank Prof.Peter structive comments or providing samples.We HOLLINGSWoI订H for his con. the manuscript.This study was the Research Fund for the Large.Scale supported by Scientific Facilities of Chinese Academy of Sciences on (Grant No.2009.LSF.GBOWS.0 1 1.the West Light Pro. gramme of the Chinese Academy of Sciences(Grant Project of Yunnan Province,China(Grant No.2008PY064).Alan FOR. 1也ST is grateful to the Gordon and Betty Moore Foun. No.9223 1l 201 1 Chase MW,Cowan RS,Hollingsworth PM,Van Den Berg C, Madrifi{in S,Petersen G,Seberg O,Jorgsensen T,Cameron KM,Carine M,Pedersen N,Hedderson TAJ,Conrad F。 Salazar GA,Richardson JE,Hollingsworth ML,Barra- TG.Kelly L,Wilkinson M.2007.A proposal for standardised protocol to barcode allland plants.Taxon 56: clough a 295-299. 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High universality of matK primers for barcoding gymnosperms 作者: 作者单位: Yan LI, Lian-Ming GAO, RAM C.POUDEL, De-Zhu Li, Alan FORREST Yan LI(Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China;Institute of Alpine Economic Plants, Yunnan Academy of Agricultural Sciences, Lijiang 674100, China) , Lian-Ming GAO(Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China), RAM C.POUDEL,De-Zhu Li(Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China;Germplasm Bank of Wild Species in Southwest China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, C), Alan FORREST(Royal Botanic Garden Edinburgh, Edinburgh EH3 5LR, UK) 刊名: 植物分类学报 JOURNAL OF SYSTEMATICS AND EVOLUTION 2011,49(3) 英文刊名: 年,卷(期): 本文链接:http://d.g.wanfangdata.com.cn/Periodical_zwflxb201103002.aspx
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