Pyromark custom assays

Bisulfite conversion of gDNA was performed (EpiTect Bisulfide Kit (Qiagen Cat. No. 59104), and regions of interest were amplified by PCR (Qiagen Pyromark Custom Assays, Qiagen PyroMark kit Cat. No. 978703) using a QuantStudio 3 real time PCR system. Pyrosequencing and analysis were performed at the Stanford University School of Medicine’s Beckman Center for Molecular and Genetic Medicine. The regions analyzed and assay numbers are shown in Table 4. Data were analyzed in SPSS (version 28, IBM).

SNPs in Pitx1 and Tbx5 transcripts were identified by Sanger sequencing in the parents of a cross between an Old Dutch Capuchine (scale-footed) and a fairy swallow (muffed) that was homozygous for the 44-kb deletion upstream of Pitx1, and PyroMark Custom Assays (Qiagen) for each SNP were designed using the manufacturer’s software. Pyrosequencing was performed on cDNA and gDNA derived from HH25 limb buds using a PyroMark Q24 instrument (Qiagen). The signal intensity ratio of feathered allele to scaled allele from cDNA samples was normalized to ratios obtained from gDNA samples from the same embryos to control for allele-specific amplification bias. Normalized ratios were analyzed by Mann-Whitney U test, and considered significant if p<(0.05 / # genes assayed) to control for multiple-testing. Each experiment was performed twice, and the results presented are representative. Primers used for each assay are listed in Supplementary file 1.