Microsatellite genotyping of Reticulitermes flavipes from Wilderness Park, Lincoln, Nebraska

Genetic markers have been widely used to understand the breeding structure and population genetic of subterranean termites. Approximately 10-20 individual workers were collected from eight sites in Wilderness Park, Lincoln, Nebraska. Sample colonies at each site were separated by at least 200 meters. One worker form each site was genotyped at eight microsatellite loci. Heads from individual workers were pulverized in 0.2 ml reaction tubes. DNA was extracted using the Gentra Systems Puregene DNA purification kit protocol, except Proteinase K solution was not added and extracted DNA was rehydrated in 80 µl of 1x TE solution. The PCR reactions were setup in 96 well plates in 15 µl reaction mixtures containing 1.5 µl of 10X of PCR buffer, 1.2 µl MgCl₂, 0.75 µl dNTPs mixture, 1.5 µl labeled dyed forward primer (WellRED Dyes), 1.5 µl reversed primer, 0.15 µl Taq DNA polymerase, 1 µl DNA template and 7.4 µl ddH2O. All loci were amplified using PCR thermal cycler program with initial denaturation step 95°C (30s), followed by 35 cycles at 95°C (30s), 54°C (30s), and 72°C (30s). The reaction was concluded with one cycle 72°C (5 min) and then cooled to and kept at 4°C until removed from the PCR thermal cycler. Samples were sequenced on a Beckman Coulter CEQ 8000 Genetic Analysis System using GenomLab^TMFragment Analysis Protocol. All eight microsatellites were polymorphic with 4 – 10 alleles per locus with the frequency of most common allele within 0.11-0.6 which indicated high level of genetic variability on a local scale.