Amplification and Sequencing
The primers used to amplify the control region in North American
specimens were initially designed to analyze the genetic diversity of
the Australian population (Rollins et al, 2011). These two primers
(svCRL1 and svPheH3) amplify the majority of the mitochondria control
region (Table S1). These primers also were used to amplify DNA from the
starling population in South Africa (Berthouly-Salazar et al., 2013).
The thermocycling conditions used here were identical to those described
in the original paper (Rollins et al, 2011). This includes a 5-min step
at 94°C, 30 cycles of 94°C for 30s, 53°C for 15s and 72°C for 30s and a
final extension step for 10 min at 72°C.
Rollins et al. (2011) also designed a series of nested primers to be
utilized in the amplification of museum specimens or highly degraded
samples (Table S1). We utilized these primers in order to sequence the
control region in four overlapping segments to eliminate possibilities
for error in the sequencing process and verify with confidence the base
in each position. All sequences were sequenced in the forward direction.
This was necessary due to a repeat region at the end of the sequence
that would not amplify in the reverse direction. All sequences were sent
to GENEWIZ, Inc (South Plainfield, NJ) for PCR cleanup via an enzymatic
purification and to be sequenced. Primer extension sequencing was
performed by GENEWIZ, Inc using Applied Biosystems BigDye version 3.1.
The reactions were then run on Applied Biosystem’s 3730xl DNA Analyzer.