Sample dissection and DNA extraction in the lab
Feather dissections were performed by taking the cells from the basal
tip of the calamus, following Morin et al. (1994). The lysis was
performed by adding 400 µl lysis buffer and 40 µl protease K, and
incubating samples at 56ºC overnight. DNA was extracted using an
automated Chemagic 360 instrument (Perkin Elmer) from a total of 957
samples corresponding to 905 feathers from fledglings, and 49 feathers
and 3 tissue samples from adult individuals. A duplicated sample
collected from a recaptured individual was included as a genotyping
control. The quality and concentration of the extracted DNA was assessed
using a NanoDrop spectrophotometer (Thermo Fisher Scientific).
Laboratory procedures were performed at the SVGM laboratories (Molecular
Genetics Veterinary Service) of the Faculty of Veterinary of the
Autonomous University of Barcelona. Some feather samples, particularly
those belonging to adults, had low DNA concentrations (below 50 ng/μL,
which is the recommended DNA concentration to ensure proper sample
genotyping according to the Thermo Fisher Scientific protocol for
QuantStudio™ OpenArray® PCR Plates). For such low-quality samples
(N=90), liquid was evaporated using a SpeedVac Vaccum Concentrator
centrifuge to increase DNA concentrations. This process was carried out
at the Centre for Research in Agricultural Genomics (CRAG) facilities.
Further details on sample quality, applied thresholds and genotyping
success related with DNA concentration are shown in Section S1 in
Supplementary Information (see also Table S1, Fig. S2 and Fig. S3).