Genetic Sex identification
We used two complementary methods to sex individuals. One procedure
involved polymerase chain reaction (PCR) using highly conserved primers
2550F and 2718R (Fridolfsson & Ellegren, 1999) to amplify the
differently sized introns of Z- and W-linked chromohelicase-DNA binding
protein 1 (CHD1) genes. PCR products were separated on a stained 1% or
2% agarose gel run in TAE buffer, revealing one band for male birds and
two for females. The other method was a bioinformatics approach where we
compared the ratio of average read depth of SNPs from ddRAD sequencing
that were aligned with the zebra finch on the Z chromosome with read
depths of SNPs on autosomes (see Toews et al., 2018). Because males have
two copies and females one copy of the Z chromosome, we would expect a
Z:autosome depth ratio of 1 in males and 0.5 in females. Using the two
approaches was confirmatory of the accuracy of each method and allowed
us to investigate and correct for any possible error if they produced
conflicting results.