PLNGene Analysis in Horses: Multiway Approach for the Investigation and Validation of Molecular Variation

In this study, a molecular characterisation of the PLN gene with whole genome sequencing (WGS) and complete transcriptome sequencing data was performed on 868 horses, supported by Sanger sequencing and the PCR-RFLP method. The PLN gene encodes phospholamban – an integral membrane protein – and while phosphorylated inhibits the SR Ca2+-ATPase (SERCA) transport of Ca2+ into reticulum in the cardiac and skeletal muscles. According to the current knowledge, we hypothesised that the presence of Single Nucleotide Polymorphisms (SNPs) in the PLN gene sequence may be related to an individual's lifestyle and would remain under selection pressure. The obtained results indicated the occurrence of 14 polymorphisms of which 7 were upstream, and 7 downstream PLN gene variants according to the EquCan3.0 reference. The mRNA sequencing confirmed the presence of 3' and 5' UTR regions belonging to the PLN transcript that was in accordance with EquCab2.0, and was missed in the current version. The comparison of two reference genomes and the validation of the NGS data allowed for the 3'UTR variant (rs1146603334) to be detected, showing differences in the genotype and allele distributions across five horse breeds. A similar genotype frequency in warmblood breeds of horses (Arabians and Thoroughbreds), compared to native and heavy horses (Polish Konik, Draft and Hucul horses), indicated that this locus was under selection pressure.