Newly emerged bee DNA amplification and genetic analysis

We genotyped 20–24 worker offspring per mated queen that emerged seven weeks post oviposition initiation.

DNA was extracted using a Chelex protocol [62]. Five closely linked microsatellite loci (Table 1) were used to infer parental genotypes [63] using Mendelian inference. Multiplex PCRs were used to amplify 10 ng of DNA in 1 μl DNA dilution buffer (Qiagen), 400 pM of each primer, 1.25x reaction buffer (Sigma), 200 μM of each dNTP, 1U of Taq-polymerase and HPLC water to a final volume of 10 μl. The temperature profile for the PCR was as follows: 5 min denaturation at 95°C, 35 cycles of 30 sec each for denaturation (95°C), annealing T_m (Table 1) and extension (72°C), followed by a final step of 5 min at 72°C. The amplified products were separated in a MegaBace automated sequencer and fragment sizes were analyzed using the Fragment Profiler software. Alleles were scored as fragment lengths in base pairs.

For each primer used to determine queen and drone genotypes from newly emerged offspring, the product size (in bp), the primer dye, the annealing temperature (T_m in°C), the pair sequences, and the allelic diversity (number of alleles per colony for 20–24 individuals genotyped ±SEM), are given [64].