# Genome-Wide DNA Methylation Analysis of Performance Variation in the 5000-m Speed Race of Yili Horses

## TL;DR

The study identifies DNA methylation patterns in Yili horses that correlate with racing performance, suggesting potential biomarkers for breeding and training.

## Contribution

The first single-base-resolution methylome of Yili horses reveals epigenetic biomarkers linked to athletic performance.

## Key findings

- Elite Yili horses show over 10,000 differentially methylated regions linked to energy and neural functions.
- Hypomethylated genes in top performers include those related to oxidative phosphorylation and neuro-muscular plasticity.
- A 'promoter hypomethylated DMR panel' could serve as non-invasive biomarkers for early performance evaluation.

## Abstract

Employing whole-genome bisulfite sequencing (WGBS), we generated the first single-base-resolution methylome of pre-race jugular blood in Yili horses (n = 6) contesting a 5000 m speed trial. High—versus average—performers exhibited > 10,000 CG-biassed differentially methylated regions (DMRs) concentrated in promoter and CpG-island landscapes. Hypomethylated DMRs in elite animals significantly targeted nuclear (PPARGC1A) and mitochondrial (ND1/ND2) oxidative phosphorylation genes together with neuro-muscular plasticity loci (axon guidance, glutamatergic synapse), indicating an epigenetically primed energy–neural axis conducive to explosive ATP provision and rapid motor unit recruitment. The data validate blood-derived DNA methylation signatures as non-invasive epigenetic biomarkers for early selection and tailored training of racing stock and provide novel molecular targets for precision breeding within the Yili horse population.

Whole-genome bisulfite sequencing (WGBS) was employed in this article to map blood DNA methylation profiles at single-base resolution in Yili horses before a 5000 m speed race, with comparative analysis of epigenetic differences between the ‘elite group’ and ‘ordinary group’ across six four-year-old stallions. The overall methylation level in the elite group was generally higher than that in the ordinary groups, with a minority of regions showing hypomethylation. For instance, the promoter regions of key metabolic and neuro-related genes exhibited significant hypomethylation. The article identified over 10,000 CG differential methylation regions (DMRs), predominantly enriched in promoter and CpG island regions, anchoring 7221 differentially methylated genes (DMGs). These DMGs were significantly enriched in key biological processes including oxidative phosphorylation, protein binding, axon guidance, glutamatergic synapses, and the Hedgehog signalling pathway. Among these, six genes—ACTN3, MSTN, FOXO1, PPARGC1A, ND1, and ND2—were selected as core candidate genes closely associated with muscle strength, energy metabolism, and stress adaptation. The study confirms that the differences in athletic ability among Yili horses have a significant epigenetic basis, with DNA methylation participating in the epigenetic regulation of athletic traits by modulating the expression of genes related to energy metabolism and neuroplasticity. The constructed “promoter hypomethylated DMR panel” holds promise for translation into non-invasive blood-based epigenetic markers for early performance evaluation and targeted breeding in racehorses. This provides a theoretical basis and molecular targets for improving equine athletic phenotypes and optimising training strategies.