Molecular analyses
DNA extraction of the collected blood samples is described in Appendix
S1. Relative erythrocyte TLs were measured in all nestlings
(n =566, average age 10.9±1.5 days) from 2002-2006 (5 cohorts from
two populations, Fig. 2) using the
real-time quantitative polymerase chain reaction (qPCR) amplification
method as described by Cawthon et al. (2002) and with modifications by
Criscuolo et al. (2009). The qPCR
method measures the ratio of the
telomere repeat copy number amount of telomere sequence to a control
single copy gene number (a non-variable “housekeeping” gene; GAPDH)
relative to a reference sample. This ratio is referred to as the
relative telomere length (TL). DNA samples were diluted with
dH2O to yield 1.67 ng/μL (corresponding to 10 ng of DNA
per well in the PCR assay) and subsequently stored at -78°C. All samples
were randomized and run in triplicates on 96-well plates, each plate
including a 2-fold serial dilution (40-2.5 ng/well) of a “standard
sample” of DNA from a single individual used to produce standard curves
for each plate, a non-target control sample, and the reference sample
(all in triplicates). Following Ringsby et al. (2015),
PCR assays were prepared using the
Absolute blue qPCR SYBR green Low Rox master mix (ThermoFisher
scientific) and run using a Stratagene Mx3005p system and analyzed using
the MxPro qPCR software (Agilent). Primers and thermal profiles are
specified in Appendix S1. Relative TLs were calculated taking plate
amplification efficiencies (all within 100±10%, mean telomere assay
efficiency was 102.2±3.8%, and 101.9±3.4% for GAPDH assays across 2x21
plates) into account using the Pfaffl (2001) method. All telomere
analyses were performed at the University of Glasgow and included 507
individuals measured by MLP (this study) and 60 individuals (a subset of
both male and female nestlings from both populations from 2002 and 2005)
measured by WB under identical laboratory conditions and using the same
reference sample (Ringsby et al., 2015).
The average of the reference
sample cycle thresholds (Ct) across all plates were 9.42±0.13 SD
(CVinterplate=1.39%,
CVintraplate=0.72%) for telomere assays and 20.58±0.09
SD (CVinterplate=0.46%,
CVintraplate=0.40%) for GAPDH assays.
Molecular sexing and pedigree
reconstruction are described in Appendix S1. Each nestling was
classified into one of three selection categories (sample sizes are
shown in Table 1): Selected (1): Both parents had been subject to
the selection regime, i.e. captured during a winter selection event and
allowed to stay in the population. Intermediate (0.5): one parent
had been subject to artificial selection. Unselected (0): No
parents had been artificially selected (i.e. accidentally not captured
during any winter selection event) and these individuals are therefore
the unselected controls. Unknown genetic parents were assumed not to
have been artificially selected (i.e. not captured or immigrated after
the selection event and thus not included in the pedigree).