Molecular Ecology Notes (2003) 3, 616 – 618 doi: 10.1046/j.1471-8286.2003.00530.x © 2003 Blackwell Publishing Ltd Blackwell Science, Ltd PRIMER NOTE Characterization of microsatellite DNA loci for the southern flying squirrel (Glaucomys volans) H. BOBBY FOKIDIS,*† NANCY A. SCHABLE,* CRIS HAGEN,* TRAVIS C. GLENN* and THOMAS S. RISCH† * Savannah River Ecology Laboratory, P.O. Drawer E, Aiken, SC 29802, USA, † Department of Biological Sciences, Arkansas State University, PO Box 599, State University, Arkansas, 72467, USA Abstract Polymerase chain reaction primers for microsatellite DNA loci (one dinucleotide, four tetranucleotide and two compound) and the conditions necessary to amplify each are described for the southern flying squirrel (Glaucomys volans). These primers were tested on 22 or more individuals from a population at the Savannah River Site in South Carolina. These microsatellite primers yielded a high allelic diversity (6 – 22 alleles/ locus), and mod- erate to high observed heterozygosities (0.318 – 0.826). Primers developed for the northern flying squirrel (Glaucomys sabrinus) were also tested for use on G. volans, with only two successful cross amplifications from the seven loci. Keywords: dinucleotide repeats, Glaucomys volans, microsatellite, polymerase chain reaction, primer, tetranucleotide repeats Received 15 May 2003; revision received 3 July 2003; accepted 30 August 2003 The southern flying squirrel ( Glaucomys volans ) is a common pine-hardwood forest inhabitant with a continuous distribution across the eastern half of the United States and northward into southern portions of Ontario and Quebec (Dolan & Carter 1977). Additionally, a few small disjunct populations are found in subtemperate montane habitats in Mexico, Guatemala, and Honduras (Diersing 1980). The widespread distribution of this species and its local abundance in areas of optimal habitat makes the southern flying squirrel a useful model for genetic-based studies of behavioural ecology, in particular, mating systems. In addition, this species inhabits areas that have experienced considerable levels of habitat fragmentation, and the limited mobility of this species in nonforested environments makes it a suitable model for studies of gene flow and genetic diversity. This paper describes seven polymorphic microsatellite loci isolated from Glaucomys volans and also tests the applicability of loci developed for the sister species, the northern flying squirrel ( Glaucomys sabrinus ) by Zittlau et al . (2000). DNA was extracted from southern flying squirrel ear tissue using Qiagen DNeasy Kits (catalogue no. 69506). Extracted DNA was enriched for (TG) 12 , (AG) 12 , (AAG) 8 , (ATC) 8 , (AAC) 8 , (AAT) 12 , (ACT) 12 , (AAAC) 8 , (AAAG) 6 , (AATC) 6 , (AATG) 6 , (ACCT) 6 , (ACAG) 6 , (ACTC) 6 , and (ACTG) 6 following a protocol modified from Hamilton et al. (1999). The detailed protocol is available from TCG. In brief, the DNA was digested with RsaI (New England Biolabs), ligated to SuperSNX linkers (SuperSNX24 Forward 5 ′ -GTTTAAGGCCTAGCTAGCAGCAGAATC- 3 ′ and SuperSNX24 Reverse 5 ′ -GATTCTGCTAGCTAG- GCCTTAAACAAAA-3 ′ ; modified from the SNX linkers of Hamilton et al . (1999)), hybridized to biotinylated microsatel- lite oligonucleotides and captured on streptavidin-coated paramagnetic beads (Dynal). Unwanted DNA was washed away and ‘captured’ DNA was recovered via polymerase chain reaction (PCR) using SuperSNX Forward primer. The product was ligated into a PCR ® 2.1 Vector and inserted into Top 10 Chemically Competent Escherichia coli (Invitrogen). The bacterial clones were screened for inserts using the β- galactosidase gene. Using M13 forward and reverse pri- mers, 192 positive colonies were amplified and 96 PCR products of 500 –1000 base pairs (bp) were sequenced using Big Dye 3.0 (Applied Biosystems) chemistry and an ABI 377-96 sequencer. Sequences from both strands were assembled and edited in sequencher 4.1 (Genecodes) and exported to ephemeris 1.0 (available at http://www.uga.edu/ Correspondence: H. B. Fokidis. Fax: 870 972 2638; E-mail: bfokidis@astate.edu