Dh5α competent cells

Total RNA was extracted from the embryos using the NucleoSpin RNA kit (MACHEREY- NAGEL). RT–PCR was performed to amplify the fragments of Gremlin1 mRNA in all bird species assessed. The primer sequences used for isolation of the fragments were Fw-5′-ATGGTCCGCACACTGTTGCC-3′ and Rv-5′-GCATTTGCCGTCACATGATGCTT-3′. The fragments were subcloned using the pGEM-T Easy Vector Systems (Promega) and DH5α competent cells (TOYOBO) and sequenced using Sanger sequencing. Sequence data were compared to a BLAST search to identify homologues of the isolated fragments. For synthesis of digoxigenin (DIG)-labelled RNA riboprobes, DNA templates with Sp6 and T7 promoters were first generated by PCR. Then, the templates were transcribed with an appropriate RNA polymerase.

Total RNA from family trio and one healthy control was extracted from whole blood using a Monarch Total RNA Miniprep kit (NEW ENGLAND BioLabs, Ipswich, MA) and subjected to reverse transcription using the PrimeScript RT reagent kit (TAKARA BIO, Kusatsu, Shiga) according to the manufacturer's protocol. We designed target‐specific primers for SLC35A2(NM_005660.2) (Figure 2c, Table S3) and 2 μl of cDNA was used for polymerase chain reaction (PCR). PCR products were separated by electrophoresis on a 3% agarose gel and extracted from gels and cloned into pGEM‐T easy vectors (Promega, Madison, WI) using Ligation high (TOYOBO, Osaka, Japan), then transfected into DH5α competent cells (TOYOBO). Competent cells were spread on LB plates containing ampicillin and incubated at 37°C overnight. We performed colony PCRs on randomly selected well‐isolated colonies, and products of the colony PCR were sequenced.