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AMARAL, A. C.

CIAMPI, A. Y.

SAMPAIO, I.

VINSON, C. C.

English

Repository Open Access to Scientific Information from Embrapa

 Molecular Ecology Notes (2005)  5 , 202–204 doi: 10.1111/j.1471-8286.2005.00876.x© 2005 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Characterization and isolation of DNA microsatellite primers for the tropical tree,  Symphonia globulifera  Linn. f. C .  C .  VINSON,*† A.  C .  AMARAL,† I .  SAMPAIO* and A.  Y .  CIAMPI†* Universidade Federal do Pará, Campus de Bragança, Rua Leandro Ribeiro Aldeia, Bragança, Pará, CEP 68600 000, Brazil,  † Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte Final, Brazilia, DF, CEP 70770 900, PoBox 02372, Brazil AbstractFive highly polimorphic microsatellite markers for  Symphonia globulifera  were developedfrom enriched repeat libraries screened for microsatellite repeats. An average of 5.8 allelesper locus and expected heterozygosity ranging from 0.649 to 0.806 were found in a sampleof 12  S. globulifera  individuals from the Tapajos National Forest, in the state of Pará in theBrazilian Amazon. These loci are being used for analysis of precise paternity testing,estimation of gene flow and parentage coefficients in a managed forest in Pará, Brazil, aspart of the Dendrogene Project. Keywords :   Amazon, microsatellites,  Symphonia globulifera , tropical tree  Received 3 September 2004; revision received 4 November 2004; accepted 4 November 2004Symphonia globulifera  Linn.f. (Clusiaceae), known in Brazilas anani, is a widespread species found in southernMexico, most of Central America, parts of the West Indies,Amazonian and Guyanan South America, and tropicalWest Africa. This species is of important economic valuefor timber extraction and for the medicinal properties ofthe latex used by the local population of the Amazonrainforest (Ribeiro  et al . 1999).We are interested in conservation and managementstrategies of timber trees such as  Symphonia globulifera  fromthe Amazon rainforest. The Dendrogene proposal is toattempt to predict the effects of forestry practice (selectivelogging) on the genetic and ecological processes whichdetermine the sustainability of the harvest of the selectedspecies (Kanashiro  et al . 2002). As part of the DendrogeneProject, we developed a battery of microsatellite markers toallow precise estimation of parentage coefficients amongtrees in the wild for mating system and gene flow studies.Protocols of construction of microsatellites markers-enriched genomic libraries described by Rafalski  et al .(1996) were used. DNA was extracted from expandedleaves of a single individual of  S. globulifera.  The DNA wasdigested with three different restriction enzymes  Sau 3AI, Mse I or  Tsp 509 (Biolabs, New England) in order to selectone that would produce a larger amount of fractionatedDNA in the range of 300 to 800 pb. Approximately 50  µ gof DNA was digested with  Tsp 509I and fragments wereseparated on a 2% agarose gel, and those from 300 to 800bp were used to construct an enriched genomic library. Thefragments were ligated to adapters  Tsp 509I restriction site.Fragments containing microsatellite sequences were selectedby hybridization with biotinylated oligonucleotides com-plementary to the repetitive poly AG/TC and recoveredby magnetic beads linked to streptavidine. Subsequentlyfragments were ligated into a pGEM-T Easy vector (Promega),transformed by electroporation in  Escherichia coli  strainXL-Blue according to Sambrook  et al . (1989). Transformantswere streaked onto agar plates (120 clones per plate) withduplicate plates on nylon membranes (Hbond N, Amersham-Pharmacia) and were regrown overnight at 37  ° C. Positiverecombinant clones were identified by hybridization withan end-labelled (AG/TC) 13  (detection of DIG-labellednucleic acids with CSPD, Boehringer Mannheim) making atotal of 225 positive clones. Transformants were dilutedin water and the fragment inserted into the plasmid wasamplified using the M13 Universal Primers by polymerasechain reaction (PCR). A total of 186 inserts were sequencedon an Applied Biosystem 377 (Perkin-Elmer) instrumentusing dye terminator fluorescent chemistry with the M13forward ( − 20) primer, according to Amersham’s protocol.The positive clones sequenced that contained repetitivesequences GA and CT, which varied in size from 9 to 25, Correspondence: C. C. Vinson. E-mail: ccvinson@yahoo.com.br P R I M E R  N O T E 203 © 2005 Blackwell Publishing Ltd,  Molecular Ecology Notes , 5, 202–204 and the position of the repetitive simple sequence wasappropriate for designing the flanking sequences asprimers were selected for designing primers pairs usingthe software  primer  3  output  (Rozen & Skaletsky 2000).Twenty primers were designed and synthesized. Micro-satellite loci were amplified using PCR in 13  µ L containing:DNA (3  η g), PCR buffer (10 m m  Tris-HCl, pH 8.3, 50 m m KCl, 1.5 m m  MgCl 2 ) (1x), primer forward and reverse(0.28  µ m  each), MgCl 2  (1.5 m m ), BSA (bovine serumalbumin, Biolabs New England) (0.25 mg/mL), dNTP (0.25m m ) and  Taq  polymerase (Invitrogen) 1.3 U. An APTC-100MJ Research Thermal Cycler was used programmed for initialdenaturation at 94  ° C for 5 min; 30 cycles of denaturationat 94  ° C for 1 min, annealing at  T a  (Table 1) for 1 min, andextension at 72  ° C for 1 min, and a final extension at 72  ° Cfor 7 min. Eletrophoresis with agarose gel 3.5% to verify theamplification of the primers. Microsatellite amplificationoccurred in 19 primers (95%).The polymorphism analysis was carried out using 12adult trees (Table 1) from the Tapajos National Forest inthe eastern Brazilian Amazon. The PCR products wereseparated on 4% denaturant poliacrilamid gels containing1 ×  TBE buffer and 7  m  Urea at 45 W, 2000 V and 100mAconstant power for 30 min according to Bassam  et al . (1991).The identification of the alleles was based on the disposi-tion of the fragments in relation to the marker 10 pb ladder.Levels of variability were detected in five loci.Data analysis for allelic frequencies for each locus wasobtained directly from genotypes observed for each indi-vidual. Number of alleles per locus, mean observed andexpected heterozygosities and Hardy–Weinberg and pair-wise equilibrium were calculated using  gda  version 1.0(Lewis & Zaykin 2001). Probabilities of paternity exclusionwere estimated using  cervus  (Marshal  et al . 1998) (Table 1).All microsatellite marker loci were highly polymorphic,with a mean of 5.8 alleles per locus. Mean observed hetero-zygosity was 0.81 and mean expected heterozygosity wasgreater than 70%. The locus Sg06 showed departure fromHardy–Weinberg expectations ( P =  3750,  P <  0.005), but nopairwise disequilibrium was detected between the fiveloci. The combined values (over all loci) for first parent andsecond parent total exclusionary power were 0.868 and0.971, respectively, with mean PIC value of 0.681.These markers will have practical application in theestimation of levels of variation between the natural andmanaged populations of  S. globulifera , as well as to helpunderstand the complexity of this species in a tropicalforest, so conservation strategies can be formulated. Acknowledgements This publication is a result of the Dendrogene Project: geneticconservation within managed forests in the Amazon (2000–04www.cpatu.embrapa.br/dendro/index.htm) executed by EmbrapaAmazônia Oriental and their partners in the bilateral cooperationbetween Brazil and the United Kingdom through the BrazilianCooperation Agency (ABC) and the Department for InternationalDevelopment (DFID).We thank Milton Kanashiro and Ian Thompsonfor their valuable comments. References Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991) Fast and sen-sitive silver staining of DNA in polyactylamide gels.  AnalyticalBiochemistry ,  196 , 80–83.Kanashiro M, Thompson IS, Yared JAG  et al.  (2002) Improvingconservation values of managed forests: the DendrogeneProject in the Brazilian Amazon.  Unasylva   53 , 25–33.Lewis PO, Zaykin D (2001)  GENETIC   DATA   ANALYSIS : Computer Programfor the Analysis of Allelic Data ,  version 1 . 0 (d16c) . Free programdistributed by the authors over the Internet from http://lewis.eeb.uconn.edu/lewishome/software.html.Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statisticalconfidence for likelihood-based paternity inference in naturalpopulations.  Molecular Ecology ,  7 , 639–655.Table 1 Information of the five microsatellite marker loci of Symphonia globulifera. Annealing temperature (Ta), number of individuals (N),total number of alleles (A), observed heterozygosity (HO), expected heterozygosity (HE), and first parent and second parent totalexclusionary power [Pr(Ex1) and Pr(Ex2)] Locus Array Primer sequences (5′–3′)Allele size range bpTa (°C) N A HO HEPr (Ex1)Pr (Ex2) Accession no.Sg 01 (CT)13 F 5′CTTCCCCTTCCTCATggATT-3′ 240–304 60 12 5 0.677 1.000 0.238 0.402 AY819036R 5′TATTggCCAAggTgAAgAgg-3′Sg 06 (CT)13 F 5′ACgACATTgggAggTTTCAG-3′ 211–281 58 12 7 0.822 0.417 0.409 0.587 AY819037(CTTT)8 R 5′CTgAAACTTAAggTgCCAAgAA-3′Sg 08 (CT)18 F 5′gTTCCAAAgCCCAAgATgAA-3′ 171–231 56 10 4 0.721 1.000 0.260 0.424 AY819038R 5′ACTgATTTCCAgCCCCTCTC-3′Sg 09 (CT)20 F 5′CCCTTTCCATTggTTCACAG-3′ 130–170 60 12 5 0.710 1.000 0.276 0.456 AY819039R 5′gAggAgggTAgCTgTTgTgg-3′Sg 10 (CT)13 F 5′ACCACAAgTTCACCCTCACC-3′ 149–205 58 12 8 0.840 0.667 0.454 0.630 AY819040(CTAT)14 R 5′gCTACgAAACCAAAgAACTAACAA-3′Mean 5.8 0.754 0.816 0.868 0.97204 P R I M E R  N O T E© 2005 Blackwell Publishing Ltd, Molecular Ecology Notes, 5, 202–204Rafalski JA, Morgante M, Powell W, Vogel JM, Tingey SV (1996)Generating and using DNA markers in plants. In: Nonmamma-lian Genomic Analysis: A Practical Guide (eds Birren B, Lai E),pp. 75–134. Academic Press, New York.Ribeiro JELS, Hopkins MJD, Vicentini A et al. (1999) Flora da Reservaducke: Guia de Identificação das Plantas Vasculares de uma Florestade Terra-firme na Amazônia Central. Instituto Nacional de Pesqui-sas da Amazônia/DFID, p. 816, Midas Printing, Manaus, Brazil.Rozen S, Skaletsky HJ (2000) primer3 on the WWW for generalusers and for biologist programmers. In: BioinformaticsMethods and Protocols: Methods in Molecular Biology (eds,Krawetz S, Misener S), pp. 365–386. Humana Press, Totowa,New Jersey.Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning:Laboratory Manual, 2nd edn, p. 7. CSHL, Cold Spring Harbor,New York.

Characterization and isolation of DNA microsatellite primers for the tropical tree, Symphonia globulifera Linn. f.

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Characterization and isolation of DNA microsatellite primers for the tropical tree, Symphonia globulifera Linn. f.

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