Molecular methods
Genomic DNA was extracted from blood using either a salt extraction
technique (Richardson et al., 2001) or, since 2013, the DNeasy blood and
tissue kit (Qiagen, Crawley, UK). Sex was determined via PCR (Griffiths,
Double, Orr, & Dawson, 1998). Individuals were genotyped at 30
polymorphic microsatellite loci (Richardson et al., 2001). Parentage
assignment was carried out using MasterBayes 2.52 (Hadfield, Richardson,
& Burke, 2006); for full details see Sparks et al. (2020). Parentage
assignment was conducted for 1,966 offspring that hatched between
1993–2018, with 89% of fathers and 86% of mothers assigned at ≥80%
accuracy. Standardised individual and maternal microsatellite
heterozygosity (H s) was calculated using the R
package Genhet 3.1 (Coulon, 2010). Two of the microsatellite loci were
excluded from this heterozygosity analysis due to pooled alleles (see
Sparks et al., 2020). Variation at exon 3 of the MHC class I loci had
previously been screened in individuals from Cousin (1,148 individuals
hatched between 1992–2009) (Richardson & Westerdahl, 2003; Wright,
2014).
Variation within the leucine-rich repeat domain of TLR3 exon 4
had previously been characterised; of the three SNPs found only one SNP
was non-synonymous and had a minor allele frequency of
>0.05 (Gilroy et al., 2017). This focal SNP is found at 198
bp in the Seychelles warbler TLR3 reference sequence (NCBI
accession number: KM657704.2), where the presence of an A or C
nucleotide caused a change of amino acid from Lysine (+ charge), to
Asparagine (polar). Variation at KM657704.2:g.198A>C
(hereafter referred to as TLR3 SNP) was genotyped in 1,647
individuals using the KASP genotyping technology by LGC Genomics,
Hertfordshire.