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.