RNA sequencing and SNP marker development
RNA sequencing produced more than 720 million pair-end reads from 16 birds (Supplementary Material S1), 14 of which were used tode-novo assemble the transcriptome containing 866.3Mb assembled into 534,815 trinity ‘genes,’ and we used 373Mb of sequence data to assemble the novel transcriptome. The resulting transcriptome was used to characterize 11,378 SNP variants, approximately one variant per 32,782 bp of reference transcriptome. We first removed variants in transcripts with no valid start codon from the identified SNPs, as such variants are likely from incomplete or non-coding transcripts. This resulted in 9,756 useable sequence variants (see Supplementary Material S2 for detailed summary statistics for SNP characterization). After optimization of multiplex groups, we retained 117 SNP loci (out of 192) to be genotyped in five multiplex groups (Supplementary Material S4).
Microsatellite and SNP marker genotyping
All individuals were successfully genotyped at all microsatellite loci. For SNP genotyping, 101 out of 117 loci were genotyped in at least 70% of the individuals (our threshold for inclusion in the analyses), and 219 of the 221 individual birds were successfully genotyped at > 90% of these 101 SNP loci and were retained for analyses. Thus, downstream analyses for the SNP loci were conducted using 101 SNP loci genotyped for 219 individuals. The final 101 SNPs consisted of 52 downstream, 11 upstream, 28 missense, and 10 synonymous variants.