Molecular sexing
We determined the sex of individuals using a polymerase chain reaction
(PCR) protocol adapted from Fridolfsson and Ellegren (1999). We used
0.25μL forward primer 2550F, 0.25μL reverse primer 2718Rand 6.25μL OneTaq® DNA Polymerase (New England BioLabs ,
Victoria, Australia) in combination with 1μL DNA for each of the 12.5μL
reactions. We used an Eppendorf® Mastercycler machine with an
annealing temperature of 48°C. To visualise the reactions, we ran the
PCRs at 100V for 30 minutes on a 1.5% agarose gel with sybrstain (Invitrogen , NSW, Australia). Males were identified through
the amplification of a single product while two products were visible
for females.