BANDEIRA, L. F.

BLANCO, A. J. V.

CIAMPI, A. Y.

COELHO, A. S. G.

PEREIRA, M. F.

English

Repository Open Access to Scientific Information from Embrapa

Molecular Ecology Resources (2008) 8, 1329–1331 doi: 10.1111/j.1755-0998.2008.02275.x© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing LtdBlackwell Publishing LtdPERMANENT GENETIC RESOURCESDevelopment of microsatellite markers in Annona crassiflora Mart., a Brazilian Cerrado fruit tree speciesM. F.  PEREIRA,* L .  F.  BANDEIRA,† A.  J .  V.  BLANCO,† A.  Y.  CIAMPI§ and A.  S .  G .  COELHO‡*Graduate Program in Agronomy (Genetics and Plant Breeding), †Graduate Program in Molecular and Cell Biology, ‡Universidade Federal de Goiás, Campus Samambaia, Goiânia, GO, 74001-970, PO Box 131, Brazil, §Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, Brazília, DF, 70770 900, PO Box 02372, BrazilAbstractAnnona crassiflora Mart. (Annonaceae) is a native fruit species of the region of BrazilianCerrado with a high agronomic potential, although without any traces of domestication. Aset of 10 microsatellite primer pairs was developed from an enriched genome library(TC13). An average of 19.3 alleles per locus was detected. Observed heterozygosityestimates were consistently lower than those obtained for gene diversity, evidencing adeparture from Hardy–Weinberg expected proportions. The reported set of markersshowed to be highly informative and constitutes a powerful tool for the development ofgenetic characterization studies in A. crassiflora.Keywords: araticum, Brazilian savannah, Cerrado, genetic diversity, molecular marker, SSRReceived 3 October 2007; revision accepted 7 April 2008Annona crassiflora Mart. belongs to the family Annonaceaeand is known as araticum in Brazil. It is one of the mosttypical native fruit tree species of the Cerrado region(Brazilian woodland-savannah) that has received greatattention from research in the Brazilian Center-West regionin face of the potential for becoming a commercial crop. Itsfruits are very much appreciated by the local populationfor consumption, either fresh or processed (ice cream,popsicles, jellies and pulp) (Almeida et al. 1998). Thespecies is also known to possess pharmaceutical propertiesdetected in treatment of tumours, aside from its bactericidaland antifungal effects (Ribeiro et al. 2000). The ripe fruitpulp is used as astringent and tonic, and the infusion ofleaves and seeds is used against syphilis, diarrhea andrheumatism (Almeida et al. 1987). As most native Cerradofruit species, A. crassiflora is still in the wild state and allcommercial exploration occurs on an entirely predatorybasis, without any degree of domestication.It is widely known that simple sequence repeat (SSR)markers are excellent tools for the development of geneticcharacterization studies of wild plant species. In this study, wedescribe a set of 10 SSR markers that showed to be highlyuseful for the development of genetic studies in A. crassiflora.An enriched genomic library for A. crassiflora was pre-pared according to the protocol of Rafalski et al. (1996). Thegenomic DNA extracted from young leaves (50 μg) wasdigested with enzyme Sau3A1 (QIAGEN) and separated in2% agarose gel. The fragments of 200–800 bp were recoveredusing the Quiaquick Gel Extraction kit (QIAGEN). Individualfragments were ligated to adaptors and hybridized withbiotin-labelled probes (TC13) and recovered using magneticbeads (streptavidin-coated magnetic beads). The DNAfragments were then cloned into the plasmid pGEM T-EasyVector (Promega) and transformed into competent Escherichiacoli XL1-Blue cells (Sambrook et al. 1989). The transformedcells were plated and incubated at 37 °C for 12 h in 1×Luria-Bertani (LB) culture medium containing Ampicillin,Xgal and IPTG (50 μg/mL ampicilin, 40 μg/μL X-gal and0.5 m IPTG). Positive clones were screened using blue/whiteselection. The recombinant bacterial clones were lysed inwater and an aliquot was used for an amplification reactionCorrespondence: A.S.G. Coelho, Fax: +55-62-35211190; E-mail: acoelho@icb.ufg.br1330 P E R M A N E N T  G E N E T I C  R E S O U R C E S© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltdaccording to the Invitrogen polymerase chain reaction(PCR) protocol. The forward and reverse M13 primers,complementary to the vector region, were used in thisreaction. The PCR products were purified using ethanolprecipitation with potassium acetate. Cloned fragmentswere sequenced from both ends using BigDye Terminatorversion 3.1 kit (Applied Biosystems). Appropriate micros-atellite and flanking regions for primer design were foundin 44 clones. Primers were designed using the programPrimer 3 (Rozen & Skaletsky 2000). Microsatellite loci wereinitially tested using DNA of 12 plants of natural popula-tions. For the PCR amplification, a final volume of 13 μL wasused containing: 4.5 ng of DNA, 0.25 μm of each forwardand reverse primers, 0.25 mm dNTPs, buffer (10 mm Tris-HClpH 8.3, 50 mm KCl), 1.5 mm MgCl2, 0.25 mg/mL BovineSerum Albumin, 1 U Taq polymerase (Invitrogen) and ultr-apure water. A thermocycler PTC-100 MJ Research wasused with the programme: 94 °C for 5 min, 30 cycles at 94 °Cfor 1 min, 1 min at primer-pair-specific annealing tempera-ture, 72 °C for 1 min and a final extension at 72 °C for 7 min.Among the 44 initially synthesized primer pairs, 10primers pairs (Table 1) were selected, based on consistencyand level of polymorphism, for a preliminary genetic char-acterization of natural A. crassiflora populations collectedin the state of Goiás, Brazil. Forward primers of each pairwere labelled with different fluorochromes (6-FAM, HEXor NED) and arranged in multiplex sets for analyses in anautomated fragment analyser (ABI-3100, Applied Biosys-tems). A mix of fluorescent (ROX) labelled fragmentssynthesized as described in Brondani & Grattapaglia (2001)was used as internal size standard. The software GeneMa-pper version 3.5 (Applied Biosystems) was used for allelecalling. Genotypes for each loci in 32 adult plants from onenatural population (17°19′25″S, 51°33′47″) were evaluated.Genetic data were analysed using the program gda (Lewis& Zaykin 2001). The results suggest that there is a high levelof genetic diversity in natural populations of A. crassiflora(Table 1). An average of 19.3 alleles per locus was estimated.The estimated observed heterozygosities (mean = 0.805)was consistently lower than gene diversities estimates(mean = 0.906), evidencing an increase of homozygosity inrelation to the expected levels under Hardy–Weinbergequilibrium. Significant departures from Hardy–Weinbergproportions were detected for seven of the 10 describedloci, suggesting that the species possibly adopts a mixedself and random mating system for reproduction. Thepresence of null alleles could not be discarded, however.Significant levels of linkage disequilibrium were notdetected between any pair of loci (Table 2). The reported setof markers showed to be highly informative and constitutesa powerful tool for the development of genetic character-ization studies in A. crassiflora.Table 1 Primer sequence, repeat motif, range of fragment size, GenBank Accession number, and genetic characterization of 10 microsatellitemarkers in Annona crassiflora Mart., screened in 32 individualsLocus Primers (5′–3′) (TC)n Size (bp)Accession no. Ta n A HO HE f P valueAcr01 F 6FAM-CGGCCTTCAAAAGGGAGATA 20 200–300 EU487524 58 29 31 0.793 0.966 0.182 0.000R CATGATTCTTCTGCTTCTGTGGAcr10 F 6FAM-TGACGAAAACGAGAAAAGCA 9 150–180 EU487525 58 29 9 0.586 0.803 0.273 0.000R ATGTCCCCAACCCAATACATAcr19 F HEX-GAGAGCTGGGAGAAGAGCAA 11 145–175 EU487526 58 29 12 0.690 0.837 0.178 0.080R AAAGCTGGGAGAGACGACACAcr20 F 6FAM-AGAGCCAGAGCCAGTGAGAC 21 170–210 EU487527 60 30 17 0.933 0.896 –0.042 0.035R TTGCCTCCATCTCTCAATCCAcr22 F 6FAM-CTGACTCGCTGGCTCTCTCT 18 180–240 EU487528 58 32 19 0.875 0.924 0.054 0.153R CTACAGCCCACATGTGCAACAcr26 F HEX-CACGACCAAGGAGAGAGGAG 15 150–180 EU487529 58 32 10 0.719 0.849 0.156 0.017R GGCAACAATCCTGACTCACAAcr33 F HEX-CAAACAGGCGATGAGACAGA 32 100–230 EU487530 58 32 22 0.750 0.927 0.193 0.000R TGGTTGGCTTTTCTCTTTCAAAcr34 F NED-GGAACAGAAGCTGTGGCATT 28 130–190 EU487531 58 31 21 0.903 0.941 0.041 0.274R CGCGCAATTCCACAATAACAcr37 F HEX-GGCAACTTCTCCCCTTTACC 24 250–360 EU487532 60 31 28 0.839 0.969 0.137 0.010R CCGGTGCCTGCTGTATATGAcr44 F NED-CAATTGCAATGGGTAGAGAGAG 26 100–160 EU487533 58 32 24 0.969 0.955 –0.015 0.651R CATCACCGCACAAAGAGAA 30 19.3 0.806 0.907 0.113 —Ta, annealing temperature (°C); n, number of individuals successfully genotyped; A, number of alleles; HO, observed heterozygosity; HE, gene diversity; f, intrapopulation fixation index; P value, significance levels associated to the deviates from HWE proportions.P E R M A N E N T  G E N E T I C  R E S O U R C E S 1331© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing LtdReferencesAlmeida SP, Proença CEB, Sano SM, Ribeiro JF (1998) Cerrado:Espécies Vegetais Úteis. Embrapa-CPAC, Planaltina, Brazil.Almeida SP, Silva JA, Ribeiro JF (1987) Aproveitamento Alimentar deEspécies Nativas do Cerrado: Araticum, Baru, Cagaita e Jatobá.Embrapa/CPAC, Planaltina, Brazil.Benjamin Y, Hocheberg Y (1995) Controlling the false discoveryrate: a practical and powerful approach to multiple testing.Journal of Royal Statistical Society, 57, 289–300.Brondani RPV, Grattapaglia D (2001) Cost-effective method tosynthesize a fluorescent Internal DNA standard for automatedfragment sizing. BioTechniques, 31, 793–800.Lewis PO, Zaykin D (2001) Genetic Data Analysis (GDA): ComputerProgram for the Analysis of Allelic Data, Version 1.0 (d16c). Freeprogram distributed by the authors over the internet from http//lewis.eeb.uconn.edu/lewishome/software.html.Rafalski JA, Morgante M, Powel W, Vogel JM, Tingei SV (1996)Generating and using DNA markers in plants. In: Analysis ofNon Mammalian Genomes a Pratical Guide (eds Birren B, Lai E),pp. 75–134. Academic Press, New York.Ribeiro JF, Brito MA, Scaloppi Junior EJ, Fonseca CE (2000) Araticum(Annona Crassiflora Mart.) Série Frutas Nativas 12. Funep, Jaboticabal,SP, Brazil.Rozen S, Skaletsky HJ (2000) Primer 3 on the WWW for generalusers and for biologist programmers. In: Bioinformatics Methodsand Protocols: Methods in Molecular Biology (ed. Krawetz SMS),pp. 365–386. Humana Press, Totowa, New Jersey.Sambrook J, Fritch EF, Maniatis T (1989) Molecular Cloning: ALaboratory Manual, 2nd edn. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York.Table 2 Significance levels (P values)obtained for the linkage disequilibriumtests for all pairs of loci. All values weredeclared nonsignificant at the 5% level,under the false discovery rate criterion(Benjamin & Hocheberg 1995)Acr10 Acr19 Acr20 Acr22 Acr26 Acr33 Acr34 Acr37 Acr44Acr01 —Acr10 1.0000Acr19 0.0726 0.6947Acr20 1.0000 0.6970 1.0000Acr22 1.0000 1.0000 1.0000 0.1995Acr26 1.0000 0.8044 1.0000 1.0000 1.0000Acr33 1.0000 1.0000 0.3309 0.1962 0.0355 1.0000Acr34 1.0000 0.0128 1.0000 1.0000 1.0000 0.1072 1.0000Acr37 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000Acr44 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

Development of microsatellite markers in Annona crasiflora Mart., a brazilian cerrado fruit tree species

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Development of microsatellite markers in Annona crasiflora Mart., a brazilian cerrado fruit tree species

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