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BRONDANI, R. P. V.

CORRÊA, R. X.

CUPERTINO, F. B.

GAIOTTO, F. A.

GRATTAPAGLIA, D.

MELO, S. C. O.

REIS, A. M. M.

English

Repository Open Access to Scientific Information from Embrapa

ORIGINAL PAPERMicrosatellite markers for Caesalpinia echinata Lam.(Brazilwood), a tree that named a countrySônia Cristina Oliveira Melo Æ Fernanda Amato Gaiotto ÆFernanda Barbosa Cupertino Æ Ronan Xavier Corrêa ÆAlessandra Maria Moreira Reis Æ Dário Grattapaglia ÆRosana Pereira Vianello BrondaniReceived: 21 November 2006 / Accepted: 8 December 2006 Springer Science+Business Media B.V. 2007Abstract Caesalpinia echinata, commonly known asPau-brasil (Brazilwood), the famous tree that named Brazilis native to the Atlantic forest. Men extensively exploited itever since discovery and colonial times due to its value as asource of red dye. As a consequence, Brazilwood is athreatened species with populations reduced to small forestfragments. Ten polymorphic microsatellite loci weredeveloped from an enriched genomic library. Using fluor-escently-labeled primers, a total of 83 alleles were foundafter analyzing a sample of 44 trees. These high geneticinformation content markers should allow detailed inves-tigations of mating systems, gene flow, population structureand paternity in natural populations.Keywords SSR  Atlantic forest  Natural population Allelic diversity  ConservationMain textCaesalpinia echinata is a species of the Leguminosaefamily native to the Atlantic forest. This red-hued treereceived the common name ‘‘Pau-brasil’’ by the Portu-guese explorers as ‘‘Pau’’ is portuguese for ‘‘wood’’, and‘‘brasil’’, derived from ‘‘brasa’’, means ‘‘ember’’. Thistree soon became the most valuable source of red dye, animportant colonial export item, to a point that it gave Brazilits name. Because of intensive exploitation naturalpopulations of C. echinata were reduced to small forestfragments (Lima et al. 2002; Lira et al. 2003). Remnantpopulations have apparently suffered the constant loss ofgenetic variability due to genetic drift, inbreeding andreduced gene flow (Lima et al. 2002; Lira et al. 2003).C. echinata is currently included in the IUCN Red List ofThreatened Species as ‘‘endangered’’ (Varty 1998).Although Brazilwood is considered an importantnational tree and valued as the best wood for musicalinstrument fabrication, especially violins (Bueno 2002),very little is known about several aspects of its ecologicaland genetic features that could aid in devising efficientstrategies for its conservation. RAPD markers have beenused for a preliminary look at the genetic structure ofBrazilwood forest fragments in Brazil (Cardoso et al.1998) revealing a significant amount of variation amongpopulations in the state of Rio de Janeiro. Maternallyinherited chloroplast microsatellites also revealed a majorstructured pattern with most of the genetic variation foundbetween geographical regions and between populationswithin regions (Lira et al. 2003). To date, however, noclear picture of the overall genetic variation for therecombining nuclear DNA has been generated due to thelack of adequate, co-dominantly inherited markers. HereS. C. O. Melo  F. A. Gaiotto (&) F. B. Cupertino  R. X. CorrêaDepartamento de Ciências Biológicas, UniversidadeEstadual de Santa Cruz, Ilheus, BA, Brazile-mail: gaiotto@uesc.brA. M. M. Reis  D. GrattapagliaUniversidade Católica de Brası́lia, Brasilia, DF, BrazilD. GrattapagliaEmbrapa Recursos Genéticos e Biotecnologia, Brasilia, DF,BrazilR. P. V. BrondaniLaboratório de Biotecnologia, Embrapa Arroz e Feijão,Goiania, GO, Brazil123Conserv GenetDOI 10.1007/s10592-006-9274-ywe report the development of a battery of ten highlyinformative nuclear microsatellite markers for C. echinata.The enriched genomic library was obtained using aprotocol described by Brondani et al. (1998). GenomicDNA from an individual tree of C. echinata was extractedfrom leaves according to Ferreira and Grattapaglia (1998).Approximately 50 lg of genomic DNA were digested withSau3AI, and fragments in the desired range of 280–600 bpwere recovered by DEAE-cellulose NA-45 membrane(Schleicher and Schuell, NY) via electrophoresis on 2%agarose gel. The fragments were attached to adapters of theSau3AI restriction site. Fragments containing AG/TCrepeats were selected by hybridization to biotinylatedoligonucleotides complementary to the repetitive sequence,and were recovered by magnetic beads linked to strepta-vidine. Fragments enriched for simple sequence repeatswere amplified by PCR, cloned in plasmid vector pGEM-T(Stratagene, CA) and transformed into chemically compe-tent E. coli cells. All recombinant colonies were picked,grown overnight at 37C, followed by plasmid DNAisolation from individual colonies through an alkaline lysisminiprep procedure. Using universal primers, DNA insertswere sequenced on a 377 automated DNA sequencer(Applied Biosystems, CA) using dye-terminator fluorescentchemistry. Primers pairs flanking the repetitive sequenceswere designed with the software ‘‘Primer 3.0’’ (Lincolnet al. 1991). Twenty-six pairs of primers were designed andscreened by silver staining detection in polyacrylamidegels. Ten loci were selected based on allelic hypervari-ability, low stuttering and ease of interpretation in a sampleof eight individual plants. The forward primers for theseten microsatellite markers were 5¢ end labeled with HEX,6-FAM or TET dyes.Microsatellites were characterized with a sample of 44adult individuals of C. echinata from a natural AtlanticRain Forest fragment of 200 ha (Serra do TeimosoReserve: 1512¢S, 3929¢W—Bahia State, Brazil), wherewere found about 727 different vascular plant species(Amorin et al. 2005). The PCR cocktail (13 ll) contained30 ng of genomic DNA, 250 lM dNTPs, PCR buffer(10 mM Tris–HCl, 50 mM KCl, 2.25 mM MgCl2 pH 8.3),2.5 lg/ll BSA (New England Biolabs), 0.2 lM of eachprimer and 1 unit of Taq DNA polymerase (Invitrogen).Amplifications were performed using the following proto-col: 96C for 2 min followed by 30 cycles of 94C for1 min, the primer specific annealing temperature Ta (seeTable 1) for 1 min, 72C for 1 min, and a final extensioncycle at 72C for 7 min. Alleles were genotyped on 377Table 1 Information of 10 loci to C. echinataLocus Primer pair sequence (5¢–3¢) Repeat motif Ta(C)Sizerange(bp)A HE HO Q I GenBankaccessionno.HWP-valueCE02 ggCAAggTgAACAgCAAC (GA)17 56 250–290 13 0.834 0.750 0.67661 0.05075 BV682861 0.0208CATACACACggCCTggTTCE07 ACTAgTCggAACTCgCA (CT)25 59 90–144 16 0.775 0.818 0.61977 0.06690 BV682862 0.2064CTgTgATTCgTCAATCTTCE09 CTCCTCCTTCTAAgTTgC (CT)24 59 140–160 8 0.675 0.985 0.44220 0.16692 BV682863 0.0000gTCATgCTCCAAgTTCTCCE11 TCTCTCTCCTCCAACTCC (TC)15 59 100–140 10 0.714 0.341 0.52099 0.17988 BV682864 0.0000CCTCTTCgTACgTTCATCCE14 CTTTATgggCCAgATCATgT (CT)3AT(CT)3 59 190–200 4 0.244 0.045 0.12226 0.59579 BV682865 0.0000gAgCCTTgAATCCgTCCACE18 CATgCAAAgCTAATCTgTg (TA)1CTGTTG (TA)2 59 178–200 3 0.574 0.568 0.32709 0.25455 BV682866 0.0021gCCATggAgAAgTgTTACCE19 CAAAgACCAAATTgAgAA (AG)18 59 220–250 8 0.602 0.364 0.41355 0.18554 BV682867 0.0000AgTgAgAgCATggACTgTCE23 gCTCCgATTATCTCCTCC (AG)11 63 100–140 5 0.692 0.386 0.45504 0.15442 BV682868 0.0000AAgACCAAAgACgTgAgCCE25 CAggATCCAACgTTAACT (AG)24 59 170–200 9 0.705 0.523 0.50972 0.12043 BV682869 0.0003ggCCACAATgTATCAgAACE26 gATCACACACACCACCTCTCT (GA)19 63 170–190 7 0.476 0.568 0.27012 0.32646 BV682870 0.0745TgCCAAggTggAgTCAAgPrimer pair sequence, repeat sequence, annealing temperature (Ta), size ranges, number of alleles/locus (A), expected heterozygosity (HE),observed heterozygosity (HO), paternity exclusion probability (Q), probability of genetic identify (I) based on 44 adult individuals genotypes, andHardy-Weinberg equilibrium probability (HW P-value)Conserv Genet123automated DNA sequencer (Applied Biosystems), usingGenescan v.3.1 and Genotyper v2.5 softwares andROX-500 Size Standard (Applied Biosystems).Genetic parameters, as numbers of alleles/locus (A),expected and observed heterozygosity (HE and HO,respectively) were estimated using Genetic Data Analysis(GDA) (Lewis and Zaykin 2000). The probability ofgenetic identity (I) and the paternity exclusion probability(Q) (Weir 1996) were also estimated. Genetic data for all10 markers were submitted to a Fisher’s exact test (10000bootstraps) to test for Hardy-Weinberg proportions andlinkage disequilibrium.All 10 microsatellite markers were highly polymorphic,producing 83 alleles in total. Number of alleles/locusvaried from 3 to 16, with an average of 8.3 (Table 1).Almost all loci, but CE07 and CE26, did not conform toHW expected genotypic frequencies with HO usually lowerthan HE in most loci. However, three loci displayed anexcess of heterozygous individuals (CE07, CE09, CE26)(Table 1). As several two-locus linkage disequilibriumtests were deemed significant, combined values of I and Qcould not be estimated for all loci (Weir 1996). However, abattery of eight microsatellite markers (CE09, CE11,CE14, CE18, CE19, CE23, CE25, and CE26), found to bein linkage equilibrium, allowed us to estimate partialcombined values of I and Q at 0.5 · 10–6 and 97%,respectively.In conclusion, this first set of microsatellite markersdeveloped for C. echinata is able to discriminate individ-uals and is adequate for parentage testing and geneticpopulation studies. It should significantly increase thepossibilities of in-depth investigation of the genetic statusof current forest fragments as well as remnant populationsin preserved areas. These markers are currently being usedto investigate genetic structure and gene flow patterns innatural populations of Caesalpinia echinata in southernBahia (Brazil), in order to elaborate sound strategies forconserving this important icon tree. Furthermore it will beinteresting to verify the picture emerging from the analysisof this set of co-dominant markers with that revealed byprevious studies (Cardoso et al. 1998; Lira et al. 2003) thatsuggested a strongly structured pattern of variation amongpopulations based on RAPD and cpSSR markers.Acknowledgements Research supported by Brazilian NationalResearch council—CNPq, Bahia State Foundation for ResearchSupport—FAPESB, and Biodiversitas Foundation. S.C.O.M andF.B.C. held CNPq fellowships.ReferencesAmorin AM, Fiaschi P, Jardim JG, Thomas WW, Clifton BC,Carvalho AMV (2005) The vascular plants of a forest fragmentin southern Bahia, Brazil. SIDA 21(3):1727–1752Brondani RPV, Brondani C, Tarchini R, Grattapaglia D (1998)Development, characterization and mapping of microsatellitemarkers in Eucalyptus grandis and E. urophylla. Theor ApplGenet 97:816–827Bueno E (2002) Nova viagem à terra do pau-brasil. In: Bueno E (ed)Pau-brasil, Axis Mundi, São Paulo, pp 19–38Cardoso MA, Provan J, Powell W, Ferreira PCG, de Oliveira DE(1998) High genetic differentiation among remnant populationsof the endangered Caesalpinia echinata Lam. (LeguminosaeCaesalpinioideae). Mol Ecol 7:601–608Ferreira ME, Grattapaglia D (1998) Introdução ao uso de marcadoresmoleculares em análise genética, 3rd. Embrapa-Cenargen,Brası́lia 220 pLewis PO, Zaykin D (2000) Genetic data analysis: computer programfor the analysis of allelic data. http:// alleyn.eeb.uconn.edu/gda.Cited 20 Sept 2006Lima HC, Lewis GP, Bueno E (2002) Pau-brasil: uma biografia. In:Bueno E (ed) Pau-brasil, Axis Mundi, São Paulo, pp 39–76Lincoln SE, Daly MJ, Lander ES (1991) ‘Primer’ software. Availablefrom E. Lander. Whitehead Institute, Cambridge, MALira CF, Cardoso SRS, Ferreira PCG et al (2003) Long-termpopulation isolation in the endangered tropical tree speciesCaesalpinia echinata Lam. revealed by chloroplast microsatel-lites. Mol Ecol 12:3219–3225Varty N (1998) Caesalpinia echinata. In: IUCN2006. 2006 IUCN redlist of threatened species. Available via http://www.iucnred-list.org of subordinate document. Cited on July15th 2006Weir BS (1996) Genetic data analysis II. Sinauer Associates,Sunderland, MAConserv Genet123

Microsatellite markers for Caesalpinia echinata Lam. (Brazilwood), a tree that named a country.

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Microsatellite markers for Caesalpinia echinata Lam. (Brazilwood), a tree that named a country.

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