# Analysis of ROH Characteristics Across Generations in Grassland-Thoroughbred Horses and Identification of Loci Associated with Athletic Traits

## TL;DR

This study analyzes the genetic makeup of Grassland-Thoroughbred racehorses to identify genes linked to athletic traits and inbreeding patterns across generations.

## Contribution

The study is the first to systematically characterize genomic diversity and identify candidate genes for athletic performance in Grassland-Thoroughbreds.

## Key findings

- Short ROHs (0.5–2 Mb) were most abundant in F1, F2, CY, and F3 generational groups.
- Ten high-frequency ROH regions were identified, annotated with 120 genes, including those related to muscle performance and energy supply.
- Inbreeding coefficients increased with each generation, with F3 showing the highest F_ROH value.

## Abstract

The Grassland-Thoroughbred is an excellent racehorse breed developed in northern China in recent years, characterized by speed, endurance, and environmental adaptability. In this study, whole-genome resequencing was conducted on different generational groups of this breed to systematically analyze the distribution of runs of homozygosity (ROHs), population structure, and inbreeding levels. A series of candidate genes related to athletic performance, such as FOXO1, PRDX2, and SLC25A15, was identified. The findings provide molecular insights into the genetic basis of this breed and offer valuable guidance for optimizing breeding strategies, thereby promoting its development and application in the racing industry.

The core objective of racehorse breeding is to enhance the speed and endurance of the horses. The Grassland-Thoroughbred is an emerging horse breed developed in northern China in recent years, characterized by excellent speed performance, enduring stamina, and strong environmental adaptability. However, research on the genetic characteristics within this breed and the genes associated with athletic performance remains relatively limited. We conducted whole-genome resequencing of Grassland-Thoroughbred F1, F2, F3, and the crossbred population (CY) and obtained a total of 4056.23 Gb of high-quality data after quality control. The single nucleotide polymorphisms (SNPs) were primarily distributed in intergenic regions, followed by intronic regions. Principal component analysis (PCA) and STRUCTURE revealed clear distinctions among the generations, with a notable overlap between CY and F3. Using the SNP dataset, we analyzed the number and length distribution patterns of runs of homozygosity (ROHs) in the genomes of different generational groups of Grassland-Thoroughbreds. Short ROHs ranging from 0.5 to 2 Mb were the most abundant, with the following distribution: F1 (85.15%) > F2 (82.92%) > CY (78.75%) > F3 (77.51%). Medium-length ROHs (2–8 Mb) and long ROHs (>8 Mb) together exhibited a similar but opposite trend. The average length of ROHs was 1.57 Mb. The inbreeding coefficients (F_ROH) among different generational groups of Grassland-Thoroughbreds were as follows: F1 (0.0942) < F2 (0.1197) < CY (0.1435) < F3 (0.1497). Through ROH island analysis, 10 high-frequency ROH regions were identified and annotated with 120 genes. Genomic regions and candidate genes associated with athletic traits—ACAD8, OPCML, PRDX2, NTM, NDUFB7, SCL25A15, FOXO1, and SLC4A10—were identified. These genes may play important roles in regulating muscle performance, mitochondrial energy supply, and learning and memory processes in horses and are closely associated with the athletic ability of the Grassland-Thoroughbred population. This study is the first to systematically characterize the genomic diversity and inbreeding dynamics of the Grassland-Thoroughbred during the breeding process. It identifies candidate genes that may influence athletic performance, thereby providing an important molecular foundation and theoretical basis for the genetic improvement and performance-based selection of this emerging breed.