Molecular Ecology Notes (2007) 7, 824–826 doi: 10.1111/j.1471-8286.2007.01716.x © 2007 Blackwell Publishing Ltd No claim to original US goverment works Blackwell Publishing Ltd PRIMER NOTE Ten microsatellite loci from Zamia integrifolia (Zamiaceae) ALAN W. MEEROW* and KYOKO NAKAMURA USDA-ARS-SHRS, National Germplasm Repository, 13601 Old Cutler Rd., Miami, Florida 33158, USA Abstract Ten microsatellite loci isolated from Zamia integrifolia are described. All 10 are polymorphic, with three to 10 alleles across 36 members of a single population from South Florida. Heterozygosities ranged from 0.139 to 0.889. Two loci depart significantly from Hardy– Weinberg equilibrium, and exhibit heterozygote deficiency. One locus pair exhibits significant linkage disequilibrium. The primers have also successfully amplified loci from Zamia portoricensis and Zamia ambliphyllidia. These loci will be utilized for population studies in the Caribbean Zamia pumila complex. Keywords: coontie, cycad, microsatellite, population genetics, simple sequence repeat, Zamia Received 17 November 2006; revision accepted 8 January 2007 The Zamia pumila (Zamiaceae) complex is a monophyletic and diploid (2n = 16) assemblage of cycad populations restricted to the West Indies and the southeastern United States, currently considered to comprise nine species (Stevenson 1987a,b; Sabato 1990; Géigel 2003). Zamia integrifolia is found from extreme southeastern Georgia southward through peninsular Florida (including the Florida Keys), sporadically in the Bahamas, and in coastal areas of north central Cuba, the Cayman Islands, and south central Puerto Rico (Stevenson 1991). We isolated microsatellite loci from a population of Z. integrifolia located on the grounds of the Montgomery Botanical Center, Miami, Florida, using a modification of the enrichment method of simple sequence repeat (SSR) marker development of Edwards et al . (1996). Genomic DNA was restricted, ligated to adaptors and amplified with polymerase chain reaction (PCR). The products were hybridized twice with biotin-labelled synthetic SSRs and isolated using streptavidin-coated beads (Dynal) in conjunction with a Dynal Magnetic Particle Concentrator. The eluted fragments were size-separated using Sepharose CL-4B SizeSeptember 400 Spun Columns (Amersham Pharmacia Biotech), amplified and cloned (using phage and plasmid vectors with M13 priming sites), and the clones screened by sequencing. Reverse sequences were obtained for those containing a repeat, and primers were designed from the flanking regions of the consensus sequence using the Prime module of the Wisconsin Package version 10.2 for UNIX (Genetics Computer Group). If successful and polymorphic, a fluorescently labelled forward primer was obtained. The 10 primer pairs (Table 1) were tested across a sample from a wild population located on our location in Miami-Dade County, Florida. Differences in allele size were detected on an ABI 3730 Genetic Analyser (Applied Biosystems) using capillary gel electrophoresis with genescan ROX-500 size standard (Applied Biosystems). PCR mix for all primers was 1 × buffer (with 15 m m MgCl 2 ), 200 μ m dNTPs, 250 n m each forward and reverse primer, 0.25 U Taq DNA polymerase (New England Biolabs), 10 ng genomic DNA template and nuclease-free distilled water to a total volume of 10 μ L. We used the following PCR program on an ABI 9700 thermocycler (Applied Biosystems), adjusting annealing temperature and number of cycles as indicated (Table 1): 2 min at 94 ° C, 35 or 38 cycles of (30 s at 94 ° C, 1 min at 58–67 ° C, 1 min at 72 ° C), 10 min at 72 ° C and 4 ° C storage. Preliminary analysis of raw microsatellite data was performed using genemapper 3.5 (Applied Biosystems). Descriptive statistics (Table 1) were generated with genalex 6 (Peakall & Smouse 2006). Tests for null alleles were conducted with microchecker (Oosterhout et al . 2004). Tests for Hardy–Weinberg equilibrium (HWE), the exact test of probability (Haldane 1954; Weir 1990; Guo & Thompson 1992), and the U -test (Rousset & Raymond 1995) for heterozygote excess or deficiency, as well as link- age disequilibrium were run with genepop 3.4 (Raymond & Rousset 1995). Correspondence: Alan W. Meerow, Fax: (305) 969-6410; E-mail: ameerow@saa.ars.usda.gov