Molecular Ecology Notes (2004) 4, 265 –267 doi: 10.1111/j.1471-8286.2004.00642.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Isolation and characterization of microsatellites in the columnar cactus: Polaskia chichipe and cross-species amplification within the Tribe Pachycereeae (Cactaceae) A. OTERO-ARNAIZ,* J. CRUSE-SANDERS,† A. CASAS* and J. L. HAMRICK‡ *Instituto de Ecologia, UNAM, Campus Morelia, Apdo. Postal 27–3 (Xangari), Morelia 58089, Michoacán, México, †Rancho Santa Ana Botanic Garden, 1500N. College Ave., Claremont, CA 91711, USA, ‡ Department of Plant Biology and Genetics, 2502 Plant Sciences, University of Georgia, Athens, GA 30602, USA Abstract Microsatellite markers were developed for the columnar cactus Polaskia chichipe from central Mexico. After an enrichment procedure and three screening steps 87% of colonies contained microsatellites. A pair of primers for 10 loci (seven polymorphic) were developed, tested and used to estimate variation in samples of 18 – 45 individuals from the Tehuacan Valley, Mexico. Alleles per locus ranged from two to eight (mean 5.28; SD 2.5). Range of expected heterozygosity values was 0.188 – 0.797 (mean 0.502; SD 0.25). These loci are par- ticularly useful for more precise evolutionary studies, such as gene flow and breeding systems, for this cactus species. Keywords: dinucleotide repeats, cactus, genetic structure, molecular markers, polymerase chain reaction, simple sequence repeat Received 26 January 2004; revision accepted 12 February 2004 Cactaceae includes approximately 1600 species native to the Western Hemisphere where they are dominant in arid and semiarid zones (Nobel 2002), and have both ecological and economic importance (Casas & Barbera 2002). There are few reports on the genetic composition of natural cacti populations in the literature. Most are estimates of allo- zyme variation in columnar cacti populations pollinated by bats (Parker & Hamrick 1992; Neel et al . 1996; Sahley 1996), and almost nothing is known about the genetic diversity of cacti with other gene dispersal mechanisms and breeding systems (Nassar et al . 2002). Species studied have high levels of genetic variation and low differentiation among populations (Hamrick et al . 2002). Microsatellite markers have not been developed previously for cactus species, but are essential for detailed evolutionary studies. Evolutionary trends in reproductive biology of Polaskia chihipe have been previously investigated (Otero-Arnaiz et al . 2003). Presently our research focuses on analysing genetic structure and gene flow among populations in rela- tion to the human management of populations. Here we report the characterization of seven polymorphic micro- satellite loci isolated from P. chichipe. DNA was extracted from flower buds following a proto- col (de la Cruz et al . 1997) adjusting amounts for extraction in tubes of 1.5 mL. A genomic library was made and enriched for the motif CA/GT following the procedure by (Kandpal et al . 1994). Total DNA was digested with Sau 3AI, and frag- ments between 400 and 1500 bp were selected, purified and ligated with T4 to Sau 3A. Fragments were amplified using Sau -L-A as primer, polymerase chain reaction (PCR) product was denatured and enriched by hybridization for the repetition (CA) using the matrix VECTREX® Avidin-D. Molecules recovered from the hybridization were amplified with Sau -L-A primer and the product purified. Cloning was conducted using the vector TOPO-TA cloning kit for sequencing (Invitrogen), and transformation in TOP10 One-shot chemically competent Escherichia coli (Invitrogen), according to the manufacturer’s protocol. Colonies were hybridized with (CA) n probe and hundreds of positive clones detected with chemioluminiscence. After three screening steps, 23 positive clones were isolated and purified. Plasmids were sequenced with the Big Dye terminator kit (Applied Biosystems) using T7 and M13 universal primers, and detected with an ABI-PRISM 3700 DNA sequencer. Correspondence: Adriana Otero Arnaiz. Fax: (52) 44 33 222719; E-mail: aotero@oikos.unam.mx