METHODS
Field sampling of Bermuda’s feral
chickens (G.
Gallus )
Field samples of fresh blood were collected on Whatman Filter cards from
chickens that were culled by the Bermudian government in 2015.
Individuals were chosen for sequencing (see below) in order to include
1) a range of sublocalities spanning the small archipelago’s main
islands and 2) a range of microhabitats including developed and
undeveloped areas. In total, n=15 males and n=6 females were selected
for sequencing. Phenotypic data was not available for the individuals
that were used for Whole Genome Sequencing, and was instead collected
from individuals sampled in the field in Bermuda during 2018 (table 2).
This consisted of body weight and comb weight measures taken post-mortem
(collected from 95 Bermudian feral individuals, 55 males and 40
females), as well as comb morphology recordings (presence of pea comb,
rose comb and duplex comb morphs), and the number of different leg
colorations present in the population (yellow and/ or grey leg colours),
taken from 134 Bermudian feral individuals. In addition, comb and body
weight, and morphological measures were also taken from 36 male and 36
female Kauai feral birds. For the Kauai birds, 25 birds were blood
sampled and sequenced, with samples once again taken from a variety of
sublocalities.
Publicly available sequence data for
domestic and wild G.
gallus
Whole-genome sequence data for domestic and Red Junglefowl contrasts
were obtained by from data published by Qanbari et al (Qanbari, Rubin et
al. 2019). We downloaded the reads from the European Nucleotide Archive
(ENA, http://ebi.ac.uk/ena), and called variants using the same workflow
as for the Bermuda samples. The accession numbers and sample labels used
are listed in Supplementary Table 10.
DNA Sampling and whole genome
sequencing
We extracted genomic DNA using the DNEasy Blood and Tissue Kit (Qiagen),
according to the manufacturer’s protocol. We sequenced 21 Bermuda
chicken samples at 30X coverage on the Illumina HiSeq X platform at
SciLifeLab, Stockholm, and 25 Kauai chicken samples on an Illumina
novoseq X6000. In the Bermuda samples, we trimmed residual adapter
sequences with Trimmomatic version 0.36 (Bolger, Lohse, & Usadel,
2014), aligned the sequences to the chicken genome (version Galgal6)
using bwa mem version 0.7.17(Li, 2013) and processed the alignments
using a workflow inspired by GATK best practices (DePristo et al., 2011;
McKenna et al. , 2010; Van der Auwera et al ., 2013),
including the use of the functions MergeSamFiles, SortSam,
BaseRecalibrator, ApplyBQSR, GenomicsDBImport and GenotypeGVCFs from
GATK version 4.3.0.0. Sequencing duplicates were removed with the
MarkDuplicates function from picard versions 2.27.5
(https://broadinstitute.github.io/picard/index.html). In the Kauai
samples, poly-G tails were trimmed using fastp (Chen, Zhou et al. 2018),
after which the workflow mentioned above was followed, but with bwa mem
version 0.7.15, GATK version 3.8.1.0 and picard version 1.118. Finally,
we filtered the resulting SNPs with the GATK VariantFiltration tool. We
retained variants that passed filters QD < 2.0, FS
> 60.0, MQ < 40.0, MQRankSum < -12.5,
and ReadPosRankSum < -8.0. In a second filtering step using
bcftools version 1.14 (Danecek, Bonfield et al. 2021), SNPs with up to
40% of missingness and a minor allele frequency larger than 5% per
population were kept. All SNPs for the selective sweep analyses were
phased using 50 iterations in Beagle 5.4 (Browning, Tian et al. 2021).