Polymorphism at trinucleotide microsatellite loci in the subterranean termite Reticulitermes flavipes

The subterranean termite Reticulitermes flavipes (Rhinotermitidae) is widespread throughout the eastern U.S.A., where it is important both as a decomposer of wood and as an economic pest. The social organization and modes of colony foundation in Reticulitermes spp. are highly flexible (reviewed by Thorne 1998). However, the cryptic nesting and foraging habits of these subterranean species has made it difficult to conduct extensive studies of their social and spatial organization. Consequently, we have little information on the relative frequencies of the alternate forms of colony organization and modes of reproduction, or how these vary in response to ecological conditions. Genetic markers have great potential for elucidating colony organization and population structure, but there have only been a few studies on Reticulitermes spp. using either allozymes To provide a sensitive tool for investigating colony and population structure, I developed microsatellite markers for R. flavipes. Termites were collected from infested logs of pine and hardwood trees at various locations in North Carolina, USA. Heads of five workers from each of five colonies were used to construct the genomic library. DNA from pooled tissue was extracted using the Wizard Genomic DNA Purification Kit (Promega) after grinding in liquid nitrogen. Library construction and screening was performed largely following the protocol of Glenn (1996). The genomic DNA was digested with Sau 3AI, and fragments 300 –700 bp were selected for cloning into pZErO-2 plasmids (Invitrogen). I transformed TOP10 F ′ cells to obtain a 20 000-clone library. Plated colonies were lifted onto nylon membranes which were probed with synthetic oligonucleotides consisting of 8–10 repeats of a trinucleotide motif rich in AT (AAT, AAC, ATC, AAG and ACT). Thirty-six positive clones were sequenced. Southern blots of plasmid DNA confirmed 36 positives, for which I obtained 23 sequences containing five or more repeats. From these sequences, 20 primer pairs were designed. Microsatellite analysis was performed largely according to the methods of Oetting et al. (1995) in which the first 19 bp of the M13 forward sequencing primer (CACGACGTTGTAAAA-CGAC) is added to the 5 ′ end of one of the specific primers in each pair. The tail was attached to the left primer in each pair, except in the case of Rf 5 –10, in which it was added to the right primer. A fluorescent labelled M13 (M13F-29/IRD 800, Li-Cor) primer was included in the polymerase chain reaction (PCR), yielding labelled product which was detected in a Li-Cor …