984 PERMANENT GENETIC RESOURCES NOTE Obbard DJ, Harris SA, Pannell JR (2006) Simple allelic-phenotype diversity and differentiation statistics for allopolyploids. Heredity, 97, 296–303. Peakall R, Smouse PE (2006) GenAlEx 6: genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes, 6, 288–295. Rahn K (1996) A phylogenetic study of the Plantaginaceae. Botanical Journal of the Linnean Society , 120, 145–198. Rønsted N, Chase MW, Albach DC, Bello MA (2002) Phylogenetic relationships within Plantago (Plantaginaceae): evidence from nuclear ribosomal ITS and plastid trnL-F sequence data. Botanical Journal of the Linnean Society, 139, 323–338. Squirrell J, Wolff K (2001) Isolation of polymorphic microsatellite loci in Plantago major and P. intermedia. Molecular Ecology Notes, 1, 179–181. doi: 10.1111/j.1755-0998.2009.02542.x © 2009 Blackwell Publishing Ltd 2545 10.1111/j.1755-0998.2009.02545.x January 2009 0 958??? 960??? Permanent Genetic Resources PERMANENT GENETIC RESOURCES NOTE PERMANENT GENETIC RESOURCES NOTE PERMANENT GENETIC RESOURCES note Isolation of microsatellite loci from the endangered plant Galium catalinense subspecies acrispum (Rubiaceae) MITCHELL E. MCGLAUGHLIN,* LYNN RILEY† and KAIUS HELENURM† *School of Biological Sciences, University of Northern Colorado, Greeley, CO 80639, USA, †Department of Biology, University of South Dakota, Vermillion, SD 57069, USA Abstract Galium catalinense subspecies acrispum (Rubiaceae) is a state-endangered perennial shrub endemic to San Clemente Island. Eight polymorphic microsatellite loci were isolated from G. catalinense ssp. acrispum. These loci show high levels of variability, averaging 6.5 alleles per locus and an expected heterozygosity of 0.550. One locus exhibited significant deviations from Hardy–Weinberg equilibrium ( P < 0.01) and one pair of loci exhibited significant linkage disequilibrium. Keywords: California Channel Islands, conservation genetics, Galium, microsatellite Received 10 October 2008; revision accepted 20 November 2008 Galium catalinense A. Gray ssp. acrispum Dempster (Rubiaceae), the San Clemente Island bedstraw, is a California endangered, perennial shrub endemic to San Clemente Island, California. This species occurs primarily on rocky outcrops of steep canyon walls but is also found on canyon bottoms and open coastal slopes; populations are often small, consisting of fewer than 30 individuals, although several of these may be found in the same canyon (Junak & Wilken 1998). It is likely that G. catalinense ssp. acrispum was decimated by extensive overgrazing by goats during the past century and by the spread of non-native plants after grazers were eliminated. Due to this recent history of overgrazing and introduced competitors on San Clemente Island, understanding the population structure, effective population size, and rates of gene flow among populations is important to managing this rare plant. Here, we report the characterization of eight polymorphic microsatellite loci isolated from G. catalinense ssp. acrispum. Genomic DNA was isolated from leaf tissue using the DNeasy Plant Mini Kit (QIAGEN). Isolation of microsatellite loci was performed following the subtractive hybridization method of Hamilton et al. (1999) with some modifications. Digested DNA was enriched for eight oligonucleotide repeats (AC) 15 , (AG) 15 , (AT) 15 , (CG) 15 , (CCG) 10 , (AAC) 10 , (AGG) 10 , and (CAC) 10 . Fragments were cloned using pBluescript II SK-Phagemid vector and the XL1-Blue MRF’ bacterial host strain (Stratagene). Colour-positive clones were screened for microsatellite regions using a membrane-based ‘dot blot’ method (T. Glenn and M. Schable, unpublished) and the Phototope chemiluminescent detection system (New England Biolabs). A total of 126 positive clones were screened for insert size by polymerase chain reaction (PCR) using an MJ Research PTC-200. The 25- μL reactions contained 1 μL template DNA, 0.8 μm each of primers T3 and T7 (Integrated DNA Technologies), 1× Themopol Reaction Buffer (New England Biolabs), 200 μm of each dNTP, and 0.2 U of Vent (exo-) DNA polymerase (New England Biolabs). Clones that exhibited a single amplified band of 400–1000 bp were cleaned using a PEG precipitation procedure and sequenced Correspondence: Mitchell McGlaughlin, Fax: (970) 351-2335; E-mail: mitchell.mcglaughlin@unco.edu