Sheep (Ovis aries) are economically one of the most important animals in the world (1), providing humans with a stable and high-quality source of animal protein and animal products such as skin, hair, and milk. Since sheep were domesticated (2), they have inhabited all parts of the world following the migration of humans. In this process, both the natural environment and artificial selection have had a profound impact on the domestication of sheep, resulting in large differences in the appearances, phenotypes, and morphological structures of sheep in different regions. For example, Dorper sheep native to South Africa and Suffolk sheep (3, 4) from the United Kingdom are famous for their fast growth rates and high meat production. The average adult sheep weight range can reach 85–120 kg. In sharp contrast, small sheep, such as Ouessant sheep and Shetland sheep (5, 6), have average adult body weights ranging from 45 to 75 kg. The growth traits of sheep (such as weight, height and length) can directly reflect the growth rates and fat growth rates of sheep and are also an important indicator of the growth performance of sheep. Therefore, understanding sheep growth traits is highly important for livestock production. At present, the common sheep varieties in China include Ujumqin sheep, Hu sheep, Xiaowei Han sheep, Tibetan sheep and others (7–10). As an excellent local variety differentiated from Mongolian sheep, Ujumqin sheep gradually developed after long-term selection and breeding and are well known for their fast growth rate and delicious meat.

In recent years, following the rapid development of biotechnology and genomics, molecular marker technology has improved. Single-nucleotide polymorphisms (SNPs) are third-generation molecular markers after restricted fragment length polymorphisms (RFLPs) and microsatellite polymorphisms (MPPs) (11). SNPs refer to DNA sequence polymorphisms caused by single nucleotide variations in the genome, including single base insertions, deletions, transitions, and transversions (12). SNPs can be classified into nonsynonymous single nucleotide polymorphisms (nsSNPs) and synonymous single nucleotide polymorphisms (sSNPs) according to their effects on gene transcription and protein translation. Nonsynonymous single nucleotide polymorphisms represent common genetic variants that alter the translated amino acid sequence, and this may affect the structure or function of the expressed protein (13). This genetic variation may lead to alterations in individual traits, such as body length and height growth traits. In this context, the results of an increasing number of studies have shown that nsSNPs have important effects on the growth traits of sheep (14, 15). By continuously mining and verifying major gene loci related to sheep economic traits, including the MSTN gene affecting sheep muscle growth and development, the TBXT gene affecting sheep tail type, and the FGF5 candidate gene related to wool length, researchers have applied these polymorphisms of major genes in production practice through gene editing and other technologies. This approach can greatly increase the economic value of sheep; for example, biallelic knockout of MSTN expression in sheep was successfully achieved using CRISPR/Cas9 gene editing. Compared with wild-type (WT) sheep, MSTN knockout sheep have greater muscle mass and muscle fiber diameters, but their meat quality and taste are not affected (16). CRISPR/Cas9-mediated deletion of the FGF5 gene not only results in the loss of its activity but also promotes the growth of sheep wool and cashmere, thereby increasing length and yield (17). These achievements have brought new optimism and opportunities for the development of animal husbandry. Nevertheless, the major gene loci involved in sheep growth traits still require research, especially with the deepening of our understanding of the sheep genome driven by biotechnology. There are still some potentially important gene loci that have not been revealed. Therefore, it is of great theoretical and practical significance to identify and verify the relevant sheep growth trait sites; it is expected that this investigation will provide a breakthrough for improving sheep production performance and economic benefits. It is important to identify potential genetic markers that affect the growth traits or reproductive characteristics of sheep and to accelerate the sheep breeding process.

In this study, resequencing data from four sheep breeds were divided into high-yield and low-yield groups according to body size, and population differentiation index (FST) analysis was used to identify genes and mutation sites related to the body size of the sheep. Based on the FST results, we performed GO and KEGG enrichment analyses for the top 5% of the candidate genes. Twenty-four nsSNPs across three candidate genes, KIAA1217, STNA1 and LTBP1, were identified. However, whether these nsSNPs have an effect in Ujumqin sheep is unclear. Therefore, we collected blood from 642 Ujumqin sheep for genotyping of the above sites, matched the genotyping results with body size data, and verified the molecular markers of the main effect growth trait sites in the genomes of Ujumqin sheep.

Against the background of natural selection, some selective intervention behaviors of humans may cause mutations in some sites of the animal body so that the genetic traits of the organism are diverse. Phenotypic alterations are often caused by functional mutations in genes that control the trait, so such mutations can serve as valid molecular markers for marker-assisted breeding (30). Among these mutations, missense mutations are considered to affect the body phenotype by affecting the mRNA expression of the related genes and the structures of the proteins. In this study, the g.24429511T > C and g.62222626C > A mutations, which are missense mutations that cause differences in growth traits, were located in exon 19 and exon 4 of the gene, respectively. Although both sites lead to changes in amino acids encoded, there are some differences between the two mutations. The g.24429511T > C missense mutation caused the encoded amino acid to change from phenylalanine to leucine, and the amino acid properties changed from polar to nonpolar after the mutation, which may have led to changes in protein properties. Further analysis revealed that mutation of this site resulted in a decrease in the minimum structural free energy of the mRNA secondary structure, an increase in stability, an increase in the proportion of α-helix and random coils, and a decrease in the proportion of β-turn and extended strands. After the missense mutation at g.62222626C > A, the encoded amino acid changed from tryptophan to leucine and from a polar amino acid to a nonpolar amino acid. However, from the perspective of protein secondary structure, the mutation at this site did not cause changes in the proportions of α-helix, extended strands, β-turn or random coils. The main reason may be that amino acids form extended strands in the secondary structure before and after mutation. The SNTA1 gene encodes a cytoplasmic peripheral membrane scaffold protein. In terms of tertiary structure, changes in the amino acids of the protein may affect the structure of the protein, thus affecting the binding of related proteins and leading to the emergence of different phenotypes.

KIAA1217 (a sickle tail protein homolog) has a curly helix region and an actin interaction domain. The proteins it encodes are necessary for normal disc development, dendritic spine morphogenetic regulation, embryonic skeletal system development by regulating cell migration, multicellular biological development, and substrate adhesion-dependent cell proliferation. Mutations in KIAA1217 are associated with malformations in the human backbone and tail vertebrae, and in mice, they affect the development of the spine, resulting in a reduced number of tail vertebrae and a characteristic short tail (31). Recently, researchers have shown that rapid evolution of the regulatory region of this gene in apes may lead to tail loss, which may be related to mutations in specific gene regulatory sequences (32). It has been confirmed that the vertebrae of sheep and humans are most similar in the thoracic and lumbar regions, although they show substantial differences in some dimensions (33). SNTA1 encodes the α1-synthetic protein, a scaffold protein that is a component of the anti-muscular dystrophin-associated protein complex (34). SNTA1 is the link between the extracellular matrix, the intracellular signaling apparatus, and the actin cytoskeleton. SNTA1 is involved in the regulation of the actin cytoskeleton and actin recombination (35). The SNTA1 signaling axis plays an important role in cytoskeletal tissue (36), and researchers have found that the SNTA1 gene is associated with amino acid and ion channel binding in different parts of bovine muscle (37). The sheep LTBP1 gene, located on chromosome 3, encodes TGF-β-binding protein 1 and is a member of the potential TGF-β-binding protein family. One indel of the LTBP1 gene was detected in four sheep breeds, and the effect of the LTBP1 gene on the growth traits of small-tailed cold sheep may be related to sex (38). Cao et al. conducted genome-wide DNA methylation sequencing in a subpopulation of Chinese Mongolian sheep and found that DNA methylation in three regions and two CpG sites in LTBP1 was significantly correlated with its RNA expression, and this gene was also identified as a potential candidate gene associated with weight variation (39). Unfortunately, 5 nsSNPSs of the LTBP1 gene were not detected in the population genotyping results of Ujumqin sheep in this study. Moreover, exploring the influence of the LTBP1 gene on the growth traits of Ujumqin sheep will also constitute a direction of follow-up research.

In recent years, following the development of biotechnology and genome research, an increasing number of SNPs related to the growth traits of livestock and poultry have been discovered. Studies have shown that the g.3148C > T polymorphism of the SIRT1 gene affects the heart circumference of Tibetan sheep, and the g.8074 T > A polymorphism of the SIRT2 gene is significantly correlated with body weight and body length (40). Cao et al. discovered OARX_76354330.1 and s64890.1 to be functional SNPs for growth traits of Hu sheep through genome-wide association analysis of body weight and identified three candidate genes related to body weight in Hu sheep (41): CAPN6, ITGA11 and SCMH1. By using genome-wide high-density SNP data (600 K) for selective scanning tests for important phenotypic traits, researchers have identified genes related to sheep body size, such as RMI1 and SCD5 (42). In the selection of sheep genotypes, it is necessary to comprehensively select SNP genotypes corresponding to the target traits; we therefore need to use different methods to identify additional SNP sites that affect sheep growth traits. Overall, this study used resequencing data from different sheep breeds to mine candidate genes and nsSNPs affecting sheep body size and verified their roles in Ujumqin sheep, thereby obtaining two molecular markers that could be applied in production practice. However, it is not yet clear whether these sites are equally useful in other sheep breeds, and this will be an important direction for our future research. Our results provide new insights into the mining of SNPs related to sheep growth and provide new genetic markers for the genetic improvement in Ujumqin sheep.

In this study, resequencing data from four sheep breeds were used to identify three genes related to the body size of sheep. Twenty-four nsSNPs across three genes were identified in Ujumqin sheep. Association analysis revealed that different genotypes of two SNP loci had differences in chest circumference, chest depth, body width and caudal breadth, and mRNA secondary structure stability was enhanced after mutation. These SNPs can be used as a molecular marker for Ujumqin sheep breeding and lay a foundation for future precise molecular breeding.

The data used in this study is sourced from the BioProject database of NCBI, numbered PRJNA624020.

The animal study was approved by in this study, blood was collected in accordance with the International Guiding Principles for Biomedical Research involving animals and approved by the Special Committee on Scientific Research and Academic Ethics of Inner Mongolia Agricultural University, responsible for the approval of Biomedical Research Ethics of Inner Mongolia Agricultural University [Approval No. (2020) 056]. No specific permissions were required for these activities, and no endangered or protected species were involved. The study was conducted in accordance with the local legislation and institutional requirements.

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