Molecular Ecology Notes (2006) 6, 692–694 doi: 10.1111/j.1471-8286.2006.01312.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Development of six microsatellite loci for black mangrove (Avicennia germinans) IVANIA CERÓN-SOUZA,* ELSIE RIVERA-OCASIO,* STEPHAN M. FUNK*† and W. OWEN MCMILLAN* * Biology Department, University of Puerto Rico — Rio Piedras, PO Box 23360, San Juan, Puerto Rico 00931-3360, † Durrell Wildlife Conservation Trust, Les-Augres-Manor, Jersey, JE35BP, UK Abstract We developed six new microsatellite loci for the black mangrove (Avicennia germinans), an important member of wetland communities worldwide. Loci showed moderate to high polymorphism and a survey of four locations [Puerto Rico (Jobos Bay and Luquillo), Mexico, French Guyana] revealed clear regional (and local) population structure. All populations were genetically distinct and the two continental populations showed much higher diversity than the two insular Puerto Rican locations. These loci complement those recently published by Nettel et al. (2005) and promise to be valuable for characterizing local and regional population dynamics in the black mangrove. Keywords: Avicennia germinans, black mangrove, microsatellites Received 18 October 2005; revision received 05 December 2005; accepted 08 January 2006 Mangrove communities are disappearing at an alarming rate and are becoming increasingly fragmented as wetlands are cleared and drained for development and agriculture (Variela et al . 2001). To better characterize the consequences of these changes and design effective management strategies, it is important to understand basic population parameters such as inbreeding, dispersal, and regional and local popu- lation boundaries for key members of the mangrove com- munity. To this aim, we developed and characterized six microsatellite markers for the black mangrove ( Avicennia germinans ), one of the most important trees of mangrove communities in the Neotropics. Markers were developed from libraries by Genetic Identification Services (www.genetic- id-services.com) following Jones et al . (2002) protocol and using pooled genomic DNA from 10 individuals collected across Puerto Rico. Genomic DNA was cut with seven restriction enzymes ( Rsa I , Hae III , Bsr- 1 , Pvu II , St I , Sc I and EcoRV ). Fragment sized 300 –750 bp were ligated with adapters, hybridized with four types of single-stranded biotinylated microsatellite probes (CA, GA, ATG, TAGA), captured using a biotinylated protocol and enriched by polymerase chain reaction (PCR). After removal of adapters by restriction with Hind III, fragments were cloned into the Hind III site of pUC19, which were electroporated into Escherichia coli strains DH5 α . Recombinant clones were PCR amplified using universal M13 primers and sequenced using ABI dye terminator chemistry and an ABI 377 sequencer. All libraries showed a high rate of enrichment (62.5– 100%) except for the TAGA library, which failed. Using the software primer 3 (Rozen & Skaletsky 1998) we designed primers flanking the microsatellite sequences for 13 clones (General Bank Accessionnos DQ240220 –DQ240232), which possessed a high number of repeats. We tested all 13 loci in five individuals using standardized PCR conditions: 0.5 ng/ μ L template DNA, 1 × QIAGEN PCR buffer, 0.2 m m of each dNTP, 0.3 μ m of each forward and reverse primers, 0.25 U QIAGEN Taq and 3.0 m m MgCl 2 . Amplifications used an initial denaturation of 3 min at 94 °C, followed by 35 cycles of denaturation for 40 s at 94 ° C, annealing for 40 s at 50 ° C and 30 s at 72 ° C, and a final extension of 4 min at 72 ° C. PCR products were visualized by electrophoresis on 1.5% agarose and staining with ethidium bromide. Six loci showed clear and consistently strong amplifications in the expected size range (Table 1). We further optimized MgCl 2 concentration and anneal- ing temperature for these six loci (Table 1). We examined polymorphisms from 15 individuals collected from four locations (Luquillo and Jobos Bay on the northern and southern coast of Puerto Rico, Mexico, French Guyana). Correspondence: Ivania Cerón-Souza, Fax: 1-787-764-3875; E-mail: ivceron@yahoo.com