Screening for polymorphism and genotyping

Screening for polymorphisms was performed using a genomic DNA panel consisting of 96 samples from 12 horse breeds: Polish Primitive Horse (PPH, n = 8), Polish Coldblood Horse (PCH, n = 8), Polish Warmblood Horse (PWH, n = 8), Arabian (ARAB, n = 8), Thoroughbred (THOR, n = 8), Welsh Pony (WELP, n = 8), Shetland Pony (SHET, n = 8), Haflinger (HAFL, n = 8), Percheron (PERCH, n = 8), Fiording (FIOR, n = 8), Silesian (SIL, n = 8), and Hucul (HUC, n = 8). The samples were derived from the collection of the Horse Genetic Markers Laboratory at Poznań University of Life Sciences (Poznań, Poland), where they were previously used for parentage analysis. For each gene studied, the two overlapping fragments of the 5’-flanking region were PCR amplified. The obtained amplicons harbored 1115 bp, 1036 bp, 1113 bp, and 1137 bp of the CSN1S1, CSN1S2, CSN2, and CSN3 genes, respectively. PCR primers were designed using Primer3 software (Koressaar and Remm 2007) and synthesized by Sigma-Aldrich (United Kingdom). PCR amplifications were conducted in a T-300 thermocycler (BioRAD, USA) using the following conditions: initial denaturation (95 °C, 5 min), 35 cycles of denaturation (95 °C, 30 s), annealing of primers (various temperatures, 45 s), elongation (72 °C, 1 min), and final extension (72 °C, 10 min). The PCR primer sequences and other important amplification details are shown as supplementary material. PCR products length and integrity were checked by electrophoresis (120 V, 30 min) in 1.5% agarose gel stained with ethidium bromide. The screening for polymorphisms and genotyping were carried out using the Sanger sequencing method preceded by the enzymatic cleaning of PCR products (using Thermosensitive Alkaline Phosphatase and Exonuclease I) from unused primers and nucleotides. The sequencing reaction with the use of a BigDye Terminator v1.1 Cycle Sequencing Kit was performed in a T-100 thermocycler (BioRAD, USA) under the following conditions: initial denaturation (95 °C, 5 min), 25 cycles of denaturation (95 °C, 30 s), primer annealing (50 °C, 10 s), and elongation (60 °C, 4 min). After filtering through a 96-well plate with Sephadex (Sigma-Aldrich, Germany), the samples were electrophoretically separated in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA). The nucleotide sequences obtained in this way were then analyzed using Lasergene SeqMan Pro (version 12.2.0) software (DNAStar, USA). Afterwards, the JASPAR database resources (Khan et al. 2018) were screened to verify whether detected polymorphic variants can alter the putative consensus sequences for transcription factors.

These PCR and direct DNA sequencing techniques were also applied to genotype 74 mares used for the association analysis (the animal group is described in detail below), for which the gene expression and milk composition traits measurements were carried out.