Genotyping Equine SLC16A1 Polymorphisms

All SNPs within SLC16A1 were detected based on RNA-seq data previously obtained from Arabian horses according to EquCab2.0 reference [22 (link), 29 (link)]. Then, the fast and less cost-effective PCR-RFLP method was designed to identify polymorphisms in exon 5 (ss#3021042926), and PCR-HRM was used to identify mutation in the 5’UTR (ss#3021042925). The details of the methods used are presented in Table 1. For 48 randomly selected samples, the both amplicons were sequenced using Sanger sequencing to confirm the results obtained. The sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosysytems, Thermo Fisher Scientific), PCR products were purified using BigDye XTerminator Purification Kit (Applied Biosysytems) and next sequenced on 3500xl Genetic Analyzer (Applied Biosysytems). DNA samples from blood and hair follicles were isolated with the use of Sherlock AX DNA Isolation kit (A&A Biotechnology, Gdynia, Poland), according to protocol. Both polymorphisms were genotyped for all horses.