Amplification and sequencing
PCR amplifications were performed using a Gene Amp 9700 PCR System (PE
Biosystems) thermal cycler. For the trn T-trn L region, we
used a reaction volume of 50 µl consisting of 26.6 µl
H2O, 5 µl of supply buffer (10x), an additional 2.5 µl
of 15 mM MgCl2, 0.2 mM of each deoxynucleoside
triphosphate, 10 µl Q-solution (Qiagen), 1.5 U Taq DNA polymerase
(QIAGEN, Hilden, Germany), 50 pmol of each primer, and approximately 20
ng of genomic DNA. Cycling conditions for trn T-trn L
region: 95°C for 3 mins; 10 cycles of 30 s at 95°C, 35 s at 56°C, and 90
s at 68°C; 35 cycles of 30 s at 95°C, 35 s at 53°C, and 90 s at 68°C;
and a final extension of 10 min at 68°C. For thendh F-rpl 32 region, PCR amplification was carried out using
the Phusion Hot Start Kit (Thermo Scientific) in a reaction volume of 30
µl consisting of 17.7 µl H2O, 6 µl of supply buffer
(10x), an additional 1.5 µl of 15 mM MgCl2, 0.2 mM of
each deoxynucleoside triphosphate, 50 pmol of each primer, and
approximately 20 ng of genomic DNA. Cycling conditions forndh F-rpl 32 region: 98°C for 3 mins; 35 cycles of 30 s at
98°C, 35 s at 58°C, and 80 s at 72°C; and a final extension of 15 min at
72°C. All PCR products were purified using the QIAquick PCR Purification
Kit (QIAGEN), following manufacturer’s instructions, and re-suspended in
28 ml warmed 1x TE buffer. Sequencing was performed on either a MegaBACE
1000 (Amersham Biosciences) or an ABI 3730 XL (Applied Biosciences)
capillary sequencer.