DNA extraction and PCR
Genomic DNA was extracted from the whole body of V. emeryisamples stored in 100% EtOH using a commercial kit (Qiagen DNeasy Blood and Tissue Kit, Hilden, Germany), according to the manufacturer’s instructions. The extracted genomic DNA was kept at -20°C until further analyses.
Kobayashi et al. (2008, 2011) revealed that the nuclear genetic relationships among sexual forms of two wing morphs (L queen, L male, S queen, and S male) are different from mitochondrial genetic relationships. The S queen was distinguished from other morphs by its nuclear genetic similarity, while the four sexual forms were divided into two groups according to wing type based on mitochondrial genetic similarity (Kobayashi et al., 2008, 2011). Therefore, mitochondrial genes were chosen to investigate the relationship between the L morph and the S morph.
The primer sets used for analyses are listed in Table 1. The primer sets targeting the three mitochondrial genes were specific for V. emeryi (exceptions were Pat for COI and 21v2 and r8v2 for COII). The PCR temperature profile was as follows: denaturation for 3 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at each annealing temperature, 1–2 min at 72°C, and a final extension step at 72°C for 5 min. To examine the infection status of Wolbachia , diagnostic PCR was performed using the Wolbachia -specific primer set WspecF, R (Werren & Windsor, 2000) at the appropriate annealing temperature (Table 1). To confirm Wolbachia infection, PCR using a fragment of the cell cycle gene FtsZ was performed using samples that presented as Wolbachia -free in the first diagnostic PCR with a positive control. Wolbachia -specific FtsZ primers were used for PCR according to the method described by Baldo et al . (2006). The Maxime PCR PreMix Kit (iNtRON Biotechnology, Seongnam, Korea) was used for each amplification along with 16 µl of distilled water, 1 µl of each primer (10 pmol) and 5 ng of template DNA.
PCR amplification was conducted using either a PTC-100 Programmable Thermal Controller (MJ Research, Inc., Waltham, MA, USA) or a PeqSTAR Universal Gradient Thermocycler (Peqlab Gmbh, Erlangen, Germany). The PCR amplicons were visualized in a 1% agarose gel dyed with TopGreen Nucleic Acid Gel Stain (Genomic Base, Seoul, Korea) and purified using a commercial kit (Qiagen QIAquick PCR Purification Kit; Hilden, Germany) prior to sequencing. In all cases, sequences were read in both directions for maximum clarity.