Molecular Ecology Notes (2007) 7, 1199–1201 doi: 10.1111/j.1471-8286.2007.01828.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Isolation and characterization of eight polymorphic microsatellite loci in the rainforest canopy tree, Syzygium sayeri (Myrtaceae) MIA J. HILLYER, SARAH L. BOULTER, ROGER L. KITCHING and JANE M. HUGHES Australian School of Environmental Studies, Griffith University, Nathan, Qld 4111, Australia Abstract Eight polymorphic microsatellite loci were isolated from the rainforest canopy tree, Syzygium sayeri, in order to study parentage and subsequently pollen dispersal among individuals in wild populations. Screening of one natural population (n = 64) mapped in a 500 × 500 m area at Cape Tribulation, north Queensland, Australia, yielded two to 11 alleles per locus with observed levels of heterozygosity ranging from 0.07 to 0.70. One locus was significantly out of Hardy–Weinberg equilibrium after Bonferroni correction for multiple tests. These loci should provide a useful tool in further understanding the dispersal patterns of this species. Keywords: enrichment, microsatellites, Syzygium sayeri Received 2 December 2006; revision accepted 13 April 2007 Syzygium sayeri (Myrtaceae) is a canopy tree of the tropical lowland rainforests of far north Queensland, Australia. This species uses a ‘hedge betting’ pollination strategy, whereby both vertebrates capable of long distance pollen dispersal (honey eaters and bats) and a diverse array of smaller, less-mobile insects are capable of successful pollination (Boulter et al . 2005). Both categories of pollen vectors contribute equally to pollination; however, the genetic implications of this differential pollen flow are not known. Genetic markers offer an important surrogate of the distance pollen is moved; therefore, we developed poly- morphic microsatellite loci in order to study the parentage and therefore pollen dispersal distance of a mapped popula- tion of S. sayeri . The construction of more than one genomic library was required to obtain sufficient variable microsatellite loci for S. sayeri . The first library (nonenriched) was constructed following the methods described in Hillyer et al . (2006). However, subsequent genomic libraries were enriched for di- and trinucleotide repeats and were constructed using the fast isolation by amplified fragment length polymorph- ism (AFLP) of sequences containing repeats or FIASCO method described by Zane et al . (2002) with some modifica- tions. Total genomic DNA was extracted from each individual using a DNeasy Plant mini kit (QIAGEN). DNA from a composite sample of four individuals (approximately 100 ng) was simultaneously digested with Mse I and ligated to Mse I AFLP adaptor (5 ′ -TACTCA- GGACTCAT-3 ′ /5 ′ -GACGATGAGTCCTGAG-3 ′ ). The subsequent digestion–ligation mixture (D–L mix) was then amplified with primer Mse I-N (5 ′ -GATGAGTCCTGAG- TAAN-3 ′ ), using polymerase chain reaction (PCR) and hybridized with a ‘pool’ of biotinylated probes [pool 1: (CA) 15 and (AG) 15 , or pool 2: (AAC) 8 (ACC) 8 (AGC) 8 , and (ACG) 8 ]. Hybridized DNA molecules were selectively captured using Streptavidin MagneSphere Paramagnet Particles (S-PMP) (Promega). Removal of nonspecific DNA occurred through a sequence of two nonstringency washes followed by four stringency washes. Between each wash, DNA was recovered by magnetic field separation for 3 min. Enriched DNA was resuspended in 50 μ L TE pH 8.0, heated for 5 min at 95 ° C, separated from the S-PMPs with magnetic field separation, then precipitated with one vol- ume isopropanol and sodium acetate (0.15 m ). Resultant pellets were washed in 70% ethanol and resuspended in 50 μ L double distilled water. One microlitre of recovered DNA was then amplified using PCR. Product yield was visualized on a 1.2% agarose gel, and ligated into PGEM-T easy vector, using T4 DNA Ligase (Promega). Ligations Correspondence: Mia J. Hillyer, Fax: +61 (0)73735 6717; E-mail: m.hillyer@griffith.edu.au