DNA methylation measurements
The MS-AFLP protocol used modifies the
standard AFLP protocol by substituting the MseI enzyme with the
methylation-sensitive isoschizomeric enzymes MspI and HpaII (New England
Biolabs). See Salmon et al., (2008) for further details on the MS-AFLP
method. Together, four types of variation can be scored: Type 1 is when
both enzymes cut at the restriction site and indicates no methylation,
Type 2 is when MspI does cut and HpaII does not cut, indicating the
restriction site has a methylated internal cytosine C; Type 3 is when
MspI does not cut and HpaII does cut indicating the restriction site has
hemi-methylation; and Type 4 is when neither enzyme cuts indicating
either both cytosines are methylated or the restriction site has mutated
(Richards, Schrey & Pigliucci, 2012) (conservatively, we treated Type 4
as missing data because the underlying methylation state cannot be
determined; for example see Richards et al., 2012).
We performed MS-AFLP following the protocol used by Richards et al.,
(2012). For the MS-AFLP analysis, DNA was extracted using the Gentra
Puregene tissue kit (Qiagen, Valencia, CA, USA) and was stored in 40 µl
of TE buffer. For both day 3 and day 11 samples, we digested
approximately 250ng of genomic DNA at 37º C for 3 h in paired reactions:
one with EcoRI and MspI, the other with EcoRI and HpaII. We immediately
followed the restriction digest with adaptor ligation with EcoRI and
MspI/HpaII adaptors at 16-20 h at 16º C (Supplementary Material Table 2,
all primer and adapter sequences). After adaptor ligation, we conducted
pre-selective PCR with EcoRI+1, MspI/HpaII+0 pre-selective primers
(Supplementary Material Table 2) at the following PCR conditions: 75º C
for 2 min; 20 cycles of 94º C for 30 s, 56º C for 30s, 75º C for 2 min,
final extension at 60º C for 30 min and 4º C hold. Following
pre-selective PCR, we conducted selective PCR by multiplexing 6-FAM
fluorescently labelled EcoRI+AGC primers with HEX fluorescently labelled
EcoRI+ACG primers and unlabeled primers HpaII/MspI+TCAT (Supplemental
Table 2) at the following PCR conditions 94º C for 2 min, 8 cycles of
94º C 30 s, 65º C 30 s 72º C 2 min (dropping the annealing temperature
1º each cycle), 31 cycles of 94º C 30 s, 56º C 30 s 72º C 2 min, final
extension of 60º C 5 min and a 4º C hold. We sent the selective PCR
products to Macrogen Facilities (South Korea) for fragment analysis on
an ABI 3130XL.
We used PEAKSCANNER v 1.0 (Applied Biosystems) to analyse resultant gel
files and define fragment sizes, and RAWGENO (Arrigo et al., 2012) to
define bands. We pooled data into two categories: methylated (Type 2 and
Type 3) or not methylated (Type 1). Throughout, we refer to a MS-AFLP
locus to indicate a particular sized band resolved in the selective PCR.
To ensure scores were consistent, we validated our MS-AFLP results by
duplicating the entire protocol for 30 random individuals. We identified
bands that consistently occurred, and we eliminated bands that were
inconsistently amplified or occurred at highly variable intensities. We
conducted all analyses using a binary haplotype-binding pattern
(methylated 1, not methylated 0) for 92 verified consistent CpG sites
between 50 and 500 base pairs for each nestling. We calculated
percentage of genome wide DNAm as the proportion of the 92 loci that
were methylated for each sample, we refer to this throughout as DNAm.