Unraveling the Molecular Mechanism of Bider Marking Formation in Dun Mongolian Horses Through Transcriptome...

(1) Background: The "Bider" marking refers to the symmetrical black stripes distributed on the shoulder blades of Dun Mongolian horses, representing an ancestral trait of significant genetic value. However, the molecular mechanisms underlying its formation remain unclear. This study aims to elucidate the molecular basis of these markings by comparing transcriptomic differences in skin tissues from variously pigmented areas of Mongolian horses' "Bider" patterns. (2) Methods: Using three Dun Mongolian horses as subjects, skin tissue samples were collected from their shoulders (dark-marked and light-marked areas), dorsal midline, and croup regions for transcriptome sequencing. Differentially expressed genes were identified based on sequencing data, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Key findings were validated through quantitative reverse transcription polymerase chain reaction (qRT-PCR). (3) Results: The sequencing yielded approximately 893 million high-quality clean reads, with an overall alignment rate exceeding 96%. A total of 140 to 775 differentially expressed genes were identified. GO enrichment analysis revealed that these genes were significantly enriched in biological processes related to pigment metabolism, skin and hair follicle development, signal transduction (including calcium and cyclic guanosine monophosphate (cGMP) signaling), and immune regulation. KEGG analysis further indicated that multiple pathways closely associated with pigment regulation, including the calcium signaling pathway, tyrosine metabolism, cyclic adenosine monophosphate (cAMP) signaling pathway, and melanoma pathway, were significantly enriched across different tissue comparison groups, suggesting their potential key roles in coat color phenotype formation. The reliability of the sequencing data was corroborated by the results of qRT-PCR validation. (4) Conclusions: This study conducted a transcriptome analysis of skin samples from various pigmented regions of the Dun Mongolian horse's Bider marking, revealing that the formation of this marking is associated with the differential expression of numerous genes and is co-regulated by multiple pigment-related signaling pathways.