Molecular Ecology Notes (2006) 6, 1219–1221 doi: 10.1111/j.1471-8286.2006.01496.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Microsatellite loci transferability from Theobroma cacao to Theobroma grandiflorum RAFAEL M. ALVES,* ALEXANDRE M. SEBBENN,† ANGELA S. ARTERO‡ and ANTONIO FIGUEIRA‡ *Embrapa Amazônia Oriental, CP 48, Belém, PA 66095-100, Brazil, †Instituto Florestal de São Paulo, CP 1322, SP 01059-970, Brazil, ‡Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, CP 96, Piracicaba, SP 13400-970, Brazil Abstract Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Forty-eight microsatellite loci developed for the congener Theobroma cacao were tested in cupuassu, and 29 (60.4%) produced robust alleles. The analyses of 216 cupuassu accessions using the 21 polymorphic microsatellite loci revealed a total of 113 alleles. The number of alleles per polymorphic locus ranged from two to 11, with an average of 5.38 alleles per locus. The average observed heterozygosity was 0.343, while the mean expected heterozygosity was 0.614. The successful transferability of T. cacao microsatellite primers to cupuassu was consistent with currently accepted phylogeny. Keywords: cacao, cocoa, cupuassu, Malvaceae sensu lato, SSR. Received 8 April 2006; revision received 9 May 2006; accepted 9 June 2006 Theobroma grandiflorum (Willd ex Spreng) Schum. (cupuassu; Sterculiaceae) is a fruit tree native to the Brazilian Amazon, valued by its seed-surrounding pulp. Cupuassu (2 n = 20) is related to Theobroma cacao L. (cacao), the species with the greatest economic importance of the genus. The putative centre of diversity of cupuassu is located at the Southeastern region of Pará state, Brazil (Cuatrecasas 1964), an area particularly threatened by deforestation. Establishing the genetic diversity of cupuassu populations is critical to define strategies for long- term conservation of this promising new crop. Therefore, we analysed the transferability of 48 microsatellite loci developed for T. cacao (Lanaud et al . 1999) to T. grandiflorum . The microsatellite loci were analysed in 216 individuals from seven populations. Samples of ∼ 150 mg of oven-dried leaf tissues were ground in liquid nitrogen, and DNA was extracted as described by Sereno et al . (2006). The ampli- fication reactions contained 15 ng of DNA; 1.5 m m MgCl 2 ; 100 μm of each dNTPs; 0.2 μm of each primer and 1.2 U Taq polymerase in 1× polymerase chain reaction (PCR) buffer (Invitrogen do Brazil) in a total volume of 13 μ L. Ampli- fications were conducted on a GeneAmp 9700 thermocycler (Applied Biosystems), programmed initially as described by Lanaud et al . (1999), but later the annealing temperature was set to decrease from 65 to 55 ° C by 1 ° C every cycle for 10 cycles, followed by 20 cycles at 55 ° C for 40 s. Amplifi- cation products were separated in denaturing sequencing gels (6% polyacrylamide; 7 m urea) ran in TBE at 55 W for 2 h, and visualized by silver staining (Creste et al . 2001). Each locus was tested for Hardy–Weinberg equilibrium, and observed number of alleles ( N A ); observed heterozygosity ( H O ); expected heterozygosity (H E ); exclusion power of the first [Pr(Ex 1 )] and second parent [Pr(Ex 2 )] were estimated using cervus (Marshall et al. 1998). A test for linkage disequilibrium was conducted using gda (Lewis & Zaykin 2002). From the 48 microsatellite loci tested, 29 (60.4%) pro- duced robust alleles, with 21 (43.8%) being polymorphic and eight (16.7%) monomorphic for the genotypes tested. The remaining 19 (39.6%) failed to amplify fragments under the various conditions tested. The analyses of 216 cupuassu accessions using 21 microsatellite loci revealed a total of 113 alleles. The number of alleles per locus ranged from two to 11, with an average of 5.38 alleles per locus (Table 1). The observed heterozygosity varied between loci from 0.014 to 0.829, with an average of 0.343. The expected heterozygosity varied between loci from 0.032 to 0.873, with an average of 0.614. The total exclusion power for identification of an unrelated candidate parent of an arbitrary offspring [Pr( Ex 1 )] when neither parents were Correspondence: A. Figueira, Av. Centenário 303, CP 96, Piracicaba, SP, 13400-970, Brazil. Fax: +55 (19) 3429 4814; E-mail: figueira@cena.usp.br