PCR and Sequencing
Three gene segments were amplified using previously described primers
for the nuclear genes Na-K-ATPase (NaKA) and elongation factor 1α (EF1)
(Wares, Pansky, Pitombo, Daglio, & Achituv, 2009). PCR reactions were
carried out in 25-µl reaction volumes containing 1X PCR buffer
(including 1.5 mM MgCl2), 0.2 mM of each dNTP, 1 µM of
each primer, 1 unit of Super-Term Taq polymerase (Hoffmann-La Roche),
and about 100 ŋg template DNA. PCR reactions were processed in an MJ
Research thermal cycler with the following thermal regime: an initial
step of 2 min at 95°C followed by 35 cycles of 0.5 min at 94°C, 0.5 min
at 57°C, and 1 min at 72°C, followed by 3 min at 72°C and then held at
15°C. PCR products were visualized on 1.5% agarose gels and sequenced
bidirectionally using the PCR primers on an ABI 377 DNA Sequencer
(Applied Biosystems, Foster City, CA) following the manufacturer’s
instructions.