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.