Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive
Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Multiplex PCR-based Detection of SNPs in RXFP2 Associated with the Oula Sheep (Tibetan Sheep, Ovis Aries) Horn Phenotype

Y
Yifan Li1,2,3
0009-0004-0441-8217
M
Mingyi Yan1,2,3
Y
Yi Chu1,2,3
C
Cui Han1,2,3
S
Sen Wu1,2,3,*
1Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, Qinghai 810016, P.R. China.
2Plateau Livestock Genetic Resources Protection and Innovative Utilization Key Laboratory of Qinghai Province, Xining, Qinghai 810016, P.R. China.
3Key Laboratory of Livestock and Poultry Genetics and Breeding on the Qinghai-Tibet Plateau (Qinghai), Ministry of Agriculture and Rural Affairs, Xining, Qinghai 810016, P.R. China.

ABSTRACT

Background: Horns, as a significant characteristic of ruminants, play an important role in their bodily structures and functions. A comprehensive analysis of the origin and evolution of horns will deepen our understanding of the biological mechanisms driving horn development. Relaxin/Insulin-like Family Peptide Receptor 2 (RXFP2) is a candidate gene associated with the development and morphology of sheep horns and a significant selection signal related to horn traits has been detected in the RXFP2 gene on chromosome 10.

Methods: To further explore the regulatory effects of RXFP2 on horn presence or absence in Oula sheep, an important local meat breed in Qinghai, this study employed multiplex PCR to detect the selection signal of the RXFP2 gene in the genomes of 50 horned Oula sheep (wild type) and 50 hornless Oula sheep (bred within the breed).

Result: We identified three single nucleotide polymorphisms (SNPs) associated with the horned phenotype in Oula sheep: one synonymous mutation in exon 14 (29512087:T>C) and two non-synonymous mutations in exon 17 (29505054:C>T and 29505096:C>T). This study provides additional evidence for the association between RXFP2 coding-region variants and horn phenotype in Oula sheep and offers potential molecular markers for future breeding applications after validation in larger and independent populations.

INTRODUCTION

The Oula sheep, a Tibetan breed native to the Qinghai-Tibet Plateau, is valued for meat and wool. It is characterized by robust physique, rapid growth, high-quality meat and strong cold adaptability (Li et al., 2025). Adult ewes weigh over 60 kg and rams average ~83.6 kg (Fig 1), demonstrating substantial meat production potential (Li et al., 2025). Grazing on natural pastures produces high-quality, nutritious mutton favored by consumers. However, horns in both sexes negatively affect carcass and pelt quality and reduce feed efficiency, hindering modern intensive breeding. Currently, no hornless Tibetan sheep breed has been established on the Qinghai-Tibet Plateau.

Fig 1: Representative images of Oula sheep.


       
Horn morphology was among the earliest traits investigated in sheep domestication (Kardos et al., 2015; Wang et al., 2019) and recent genomic advances have improved our ability to study such traits (Woolley et al., 2023). SNP-based marker screening and high-throughput sequencing have facilitated the identification of candidate loci and functional genes for economically important traits in Tibetan sheep (Metta et al., 2020; Sun et al., 2023, 2024). The genetic mechanisms underlying horn presence/absence are complex (Duijvesteijn et al., 2018). Early studies hypothesized three alleles at the autosomal Ho locus regulate hornlessness  (Wang et al., 2019). The “hornless locus” was mapped to chromosome 10 (Montgomery et al., 1996) and a hybrid merino × romney population narrowed it to a 50 kb region, where a 17-marker haplotype predicts horn phenotype with ~97% accuracy (Li et al., 2020). Genome-wide association studies (GWAS) identified a significant SNP near RXFP2 on chromosome 10 linked to polledness in Merino sheep (Dominik et al., 2012; Kijas et al., 2012), with additional evidence from Tibetan sheep (Tian et al., 2024). In Soay sheep, this locus also contains determinants for horn length and circumference (Beraldi et al., 2006) and RXFP2 was confirmed as a key candidate gene (Gao et al., 2025; Johnston et al., 2010). Recent discoveries have uncovered SNPs in RXFP2 that are closely associated with various horn types in sheep, suggesting a critical role in the regulation of horn development. However, similar findings have not been replicated in other wild populations. For instance, a study screening molecular markers associated with hornless traits in Qira black sheep identified several potential genetic markers, further emphasizing the genetic complexity of horn development across different sheep breeds (Cao et al., 2021; Zhou et al., 2025). Transcriptome analysis revealed RXFP2 upregulation in horn buds and identified other candidate genes including Foxl2 and Tnn (Luan et al., 2023). Recent studies have also highlighted the involvement of neural cells in horn bud initiation (Li et al., 2025) and the regulatory role of Fgf2 in sheep horn development (Liu et al., 2025).
       
A 1.8-kb insertion in the RXFP2 3′UTR was associated with polledness in European breeds (Johnston et al., 2013; Wiedemar and Drögemüller, 2015), but this association was not consistent across all breeds (Pan et al., 2018). GWAS of Altay sheep found no significant loci and the 1.8-kb insertion was not associated with hornlessness (Deng et al., 2020). Resequencing analysis of Hu sheep identified a signal near RXFP2 on chromosome 10 (Johnston et al., 2013). Whole-genome sequencing of Chinese sheep suggested RXFP2’s role in unique horn phenotypes (Pan et al., 2018). Global analysis of copy number variations (CNVs) in Chinese indigenous fine-wool sheep populations revealed several genomic regions associated with horn development (Yuan et al., 2021), reflecting the complex inheritance of horn traits (Hu et al., 2019).
       
However, most previous studies have focused on genomic region discovery rather than detailed characterization of functional coding variants in RXFP2 associated with horn phenotype in indigenous breeds. Despite extensive investigation of RXFP2 in several breeds, its coding-region polymorphisms have not been systematically characterized in Oula sheep.
       
Notably, the association between RXFP2 and horn phenotype is not universal across breeds: the 1.78-kb insertion in the 3′UTR does not segregate with horn status in breeds with variable horn status (Lühken et al., 2016) and a 1.8-kb insertion in the same region was unrelated to polledness in Altay sheep (He et al., 2016). These findings highlight the need for breed-specific investigations of RXFP2 variants. Furthermore, previous studies have mainly focused on locus discovery, whereas rapid and cost-effective molecular assays for routine breeding remain limited. Therefore, we aimed to identify coding-region SNPs associated with horn phenotype in Oula sheep and establish a multiplex PCR-based genotyping method for future marker-assisted breeding.

MATERIALS AND METHODS

Sample collection and DNA isolation
 
Animals were classified by external horn phenotype: horned individuals had fully developed horns; hornless individuals had horn length <0.5 cm and no visible horn structures; intermediate or scurs were excluded. Sheep were from a purebred population maintained by the Henan County Hornless Oula Sheep Breeding Demonstration Base in Qinghai Province. By 2023, our team had established 2 core and 4 demonstration flocks, with 1,138 hornless ewes (826 core + 312 replacement) and 57 hornless rams. All animals had clear phenotypic documentation and were adult ewes at 2 years of age. Initially, 100 sheep (50 horned, 50 hornless) were selected; after quality control (QC), reliable data were obtained from 48 horned and 46 hornless individuals, with balanced phenotype groups for comparative analyses. Blood samples were collected from the jugular vein and DNA was extracted using a rapid kit (Sangon Biotech, Shanghai) following the manufacturer’s protocol. DNA quality was assessed using a MaestroNano (MaestroGEN, USA). All animal procedures were approved by the Animal Care and Use Committee of the Qinghai Academy of Animal Husbandry and Veterinary Science (approval no. 2025-QHMKY-004) and conducted between 2023 and 2025.
 
Primer design
 
Primers were designed based on the 18 exon sequences of the RXFP2 gene available in the GeneBank database (NCBI reference sequence: NC_056063.1) [https://www.ncbi.nlm.nih.gov/] and designed using the Prime tool (Table 1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

Table 1: Primers for amplifying the 18 exons of the RXFP2 gene.


 
PCR amplification
 
A primer pool covering 18 exons was designed and tested using a two-step PCR protocol. The first PCR mixture (25 μl) contained 2 μl DNA template (10 ng/μl), 1 μl upstream primer pool (10 μM), 1 μl downstream primer pool (10 μM) and 15 μl 2× Kapa HiFi PCR ready mix. PCR conditions: 98°C for 3 min; 8 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; then 25 cycles of 98°C for 30 s, 66°C for 30 s, 72°C for 30 s; final extension at 72°C for 5 min. Products were verified on 1% agarose gel, purified with AMPure XP beads and used for second-round PCR to generate libraries. The second PCR (30 μl) contained 2 μl DNA template, 1 μl universal P7 primer (with molecular tag, 10 μM), 1 μl universal P5 primer (10 μM) and 15 μl 2× PCR ready mix. Conditions: 98°C for 5 min; 5 cycles of 94°C for 30 s, 55°C for 20 s, 72°C for 30 s; final extension at 72°C for 5 min. Products were purified, pooled and sequenced on a HiSeq XTen (Illumina, San Diego, CA). The data have been deposited in the CNGB Sequence Archive (CNSA) with accession number CNP0005902. (DOI: 10.26036/CNP0005902; available at: https://doi.org/10.26036/CNP0005902).
 
Data quality control and genotyping analysis
 
QC was performed in two steps: (1) removal of junction sequences using cutadapt (V.1.2.1, https://cutadapt. readthedocs.io/en/stable/); (2) removal of bases with quality score <20 using PRINSEQ-lite (V.0.20.3, https://sourceforge.net/projects/prinseq/files/). QC-passed sequences were aligned to the reference genome using BWA (V.0.7.13-r1126, https://bio-bwa.sourceforge.net/) with default parameters. Genotype data were generated using samtools (V.0.1.18, https://samtools.sourceforge.net/), gene annotation using annovar (https://annovar. openbioinformatics.org/en/latest/; NCBI reference: NC_056063.1) and haplotype analysis using SHEsis (https://github.com/celaoforever/SHEsisPlus).

RESULTS AND DISCUSSION

Validation and mutation analysis of coding-region SNPs in the RXFP2 using multiplex PCR
 
Using multiplex PCR, three mutations were identified in the coding region of RXFP2 (Table 2). The first mutation, in exon 14, is a synonymous mutation (A→G, proline unchanged). The second and third mutations, both in exon 17, are non-synonymous: G→A (E→K) and G→A (V→M).

Table 2: Mutations identified in the exonic regions of the RXFP2 gene.


       
The three SNP loci were identified at chromosomal positions 29512087 (T>C), 29505054 (C>T) and 29505096 (C>T) (Fig 2).

Fig 2: SNP mutation details.


 
Genetic diversity and correlation analysis of the RXFP2 gene
 
For the first mutation (29512087T>C), genotype frequencies were TT (21.28%), TC (29.79%) and CC (48.93%), with allele frequencies T (36.17%) and C (63.83%). Phenotype frequencies for TT, TC and CC were 100% hornless, 92.86% hornless/7.14% horned and 100% horned, respectively. For the second SNP (29505054C>T), the genotype frequencies were CC (21.28%), CT (30.85%) and TT (48.87%), with allele frequencies C (36.70%) and T (63.30%). The homozygous CC genotype was exclusively associated with the hornless phenotype, while the homozygous TT genotype was exclusively associated with the horned phenotype; heterozygous CT individuals showed a predominantly hornless phenotype (89.66% hornless vs. 10.34% horned). For the third SNP (29505096C>T), genotype frequencies were CC (22.34%), CT (36.17%) and TT (41.49%), with allele frequencies C (40.43%) and T (59.57%). Again, homozygous CC was exclusively hornless, homozygous TT exclusively horned and heterozygous CT individuals were predominantly hornless (73.53% hornless vs. 26.47% horned) (Table 3).

Table 3: Genotype distribution of SNPs in the coding region of the RXFP2 gene.


       
Haplotype analysis using SHEsis identified three combined haplotypes (probability > 0.03). Their frequencies in horned vs. hornless sheep were 6.3% vs. 1.1%, 90.6% vs. 27.2% and 3.1% vs. 71.7%, respectively (Table 4).

Table 4: Cardinality test for SNPs in the coding region of the RXFP2 gene.


       
Strong linkage disequilibrium (LD) was observed among the three SNPs (positions: 29,512,087; 29,505,096; and 29,505,054), with D′ and r2 approaching 1, indicating complete linkage disequilibrium (Fig 3, Table 5).

Fig 3: LD correlation diagram between SNPs.



Table 5: Analysis of linkage disequilibrium between SNP loci.


       
RXFP2, also known as leucine-rich G protein-coupled receptor 8 (LGR8), belongs to the G protein-coupled receptor (GPCR) family and is the specific receptor for insulin-like factor 3 (INSL-3) in the body (Duan et al., 2016; Feng et al., 2009). RXFP2 is a candidate gene associated with horn type in sheep. Beyond its well-characterized role in horn development and morphology, RXFP2 also has other physiological functions. For instance, a study investigated RXFP2 polymorphisms in Indian sheep and found no association with cryptorchidism, highlighting its functional diversity beyond horn traits (Kumar et al., 2019).RXFP2 is expressed in horn soft tissue and periosteum, with expression negatively correlated with horn size (Johnston et al., 2013). Recent studies have highlighted the complex tissue-specific expression and regulatory functions of RXFP2 in horn tissue, though it may not be the sole determinant of horn traits (Yang et al., 2025). Several GWAS have reported that RXFP2 is associated with horn presence but is not directly involved in horn development (Kijas et al., 2012).
       
Although previous GWAS and sequencing studies have identified RXFP2 as a major candidate gene for sheep horn phenotype, these approaches primarily provide genomic association rather than practical genotyping strategies. Here, we characterized coding-region SNPs in RXFP2 in Oula sheep and developed a multiplex PCR-based detection approach. Unlike previous genome-wide studies, we focused on specific coding-region variants, including two non-synonymous mutations and established a genotyping strategy for horn-associated alleles. Thus, this study translates previous genetic findings into a cost-effective and rapid genotyping tool that can be used as part of marker-assisted selection programs in Oula sheep, with the caveat that heterozygous individuals exhibit incomplete phenotypic penetrance and thus these markers should be interpreted alongside other phenotypic or genomic information. Validation in larger, independent populations is ongoing.
       
In the preliminary stage, horned and hornless Oula sheep populations were identified by whole-genome resequencing. Previous studies have shown that the 3′ UTR insertion in RXFP2 is associated with polledness in multiple sheep breeds (Wiedemar and Drögemüller, 2015). A strong selection signal was detected in the middle and downstream regions of RXFP2, confirming it as a major candidate gene for horn presence/absence in Oula sheep. Ovine RXFP2 encodes 763 amino acids and comprises 18 exons (Wang et al., 2014). GWAS by (Li et al., 2021) identified RXFP2 as significantly associated with horned and hornless phenotypes in sheep. A subsequent selective sweep analysis of the Manhattan plot on chromosome 10 revealed that the allele frequency distribution of a non-synonymous SNP downstream of RXFP2 across multiple breeds was consistent with our findings (Li et al., 2020). Among the three high-frequency SNPs, 29512087T>C is synonymous, while the other two are non-synonymous.
       
The two non-synonymous variants in exon 17 resulted in amino acid substitutions (p.V653M and p.E667K) in RXFP2. A recent CRISPR/Cas9 partial knockout study in sheep showed that RXFP2 expression was significantly reduced in horn buds, but horn growth and morphology were unaffected; however, the knockout sheep exhibited unilateral cryptorchidism (Gao et al., 2025). This finding supports that RXFP2 is involved in horn phenotype determination rather than directly participating in horn development and highlights the functional relevance of its coding-region variants in horn phenotype regulation. Although the functional consequences of these substitutions require further validation, changes in amino acid properties may influence protein conformation, receptor stability, or downstream signaling. Thus, these variants represent potential functional candidates for RXFP2 variation and horn phenotype in Oula sheep.
       
Similar coding-region polymorphisms were reported in Tan sheep, where resequencing of all 18 exons of RXFP2 identified multiple exonic SNPs, including a synonymous variant (p.P375, c.1125A>G) with distinct genotypes between horned and hornless individuals (Wang et al., 2014). Although synonymous mutations do not alter the amino acid sequence, accumulating evidence shows that they regulate gene expression through mRNA stability, codon usage bias, splicing and RNA modifications such as N6-methyladenosine (m6A). Recent studies further provided evidence that a single synonymous mutation can modulate phenotypic traits via epitranscriptomic regulation involving m6A modification and mRNA structural conformation (Zhou et al., 2025). These findings highlight the functional significance of synonymous variants such as p.P375 in RXFP2. Together with our Oula sheep data, this suggests that RXFP2 coding-region variation may contribute to horn phenotype diversity across breeds. Notably, allele frequencies at p.P375 in Tan sheep differ from those at the three SNPs in our Oula population (major allele frequencies: 36.17%–63.83%), reflecting population-specific selection or genetic backgrounds. A strong selection signal at RXFP2 has also been reported in Merino sheep, with a downstream non-synonymous SNP showing distinct allele frequencies between horned and polled populations (Dominik et al., 2012;  Li et al., 2020). In Tibetan sheep, GWAS have identified multiple SNPs in RXFP2 for horn length and base circumference, with allele frequencies varying across horn phenotypes (Hu et al., 2026; Tian et al., 2024). These cross-breed comparisons highlight the breed-specific genetic architecture of horn traits and suggest that RXFP2 variants have been subject to different selection pressures across populations. Moreover, a recent targeted sequencing study demonstrated that different RXFP2 variants exhibit breed-specific effects on horn morphology and polledness, supporting that the genetic architecture of horn traits is more complex than a single causative mutation (Hu et al., 2026). In addition, although the hornless trait is a well-known dominant trait, RXFP2 heterozygotes in our Oula population did not fully exhibit the hornless phenotype, suggesting that other regulatory effects may be involved (Wang et al., 2019), especially in our Oula sheep population and selection sweeps around RXFP2 associated with various horn traits further support a polygenic model for horn phenotype regulation (Guo et al., 2021).
       
PIC is often associated with within population genetic diversity (Sun et al., 2007), PIC values (0.25-0.5) indicated moderate polymorphism in Oula sheep, suggesting low genetic variation and selection potential. D′ and r2 values approached 1, indicating strong LD among the three SNPs. Homozygotes showed complete phenotype concordance at each mutation site, while heterozygotes predicted hornless phenotype with 73.53% to 92.86% accuracy. Haplotype analysis revealed three haplotypes with inheritance probability >3%; CTT showed the highest predictive probability for horned (90.6%), whereas TCC showed the highest for hornless (71.7%).
       
In Oula sheep, RXFP2 was identified as a major candidate gene for horn presence/absence. Three key SNPs in coding regions were significantly associated with horn phenotype in both horned and hornless populations and genotype identification is essential for determining horn status. These findings provide useful genetic information for understanding horn phenotype variation in Oula sheep and lay the foundation for further validation of RXFP2 variants in breeding programs.

CONCLUSION

In this study, multiplex PCR was used to genotype coding-region variants of the RXFP2 gene in 94 Oula sheep with distinct horn phenotypes. Three SNPs were identified: one synonymous mutation in exon 14 (29512087T>C) and two non-synonymous mutations in exon 17 (29505054C>T and 29505096C>T). Homozygous genotypes at all three loci showed complete concordance with horn phenotype, whereas heterozygotes displayed predominantly hornless phenotypes with 73.53%–92.86% accuracy. Haplotype analysis revealed that the CTT haplotype was strongly associated with the horned phenotype, while TCC was associated with the hornless phenotype. Strong linkage disequilibrium was observed among the three SNPs. These findings confirm that RXFP2 coding-region variants are associated with horn phenotype in Oula sheep and provide candidate molecular markers for marker-assisted selection after validation in larger, independent populations.

ACKNOWLEDGEMENT

The present study was supported by the Key Research and Transformation Project of Qinghai Province (Grant No. 2024-NK-104), entitled “Cultivation of New Variety of Hornless Oula Sheep and Integration Demonstration of Efficient Production Technologies.”
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Author contributions
 
M.Y. Yan and S. Wu conceived and designed the study. Y.F. Li performed the experiments. Y.F. Li and Y. Chu analyzed the data and wrote the manuscript. C. Han provided reagents, materials and analysis tools. All authors reviewed and approved the final manuscript.
 
Data availability
 
The sequencing data generated in this study have been deposited in the CNGB Sequence Archive (CNSA) of CNGBdb under accession number CNP0005902 and are publicly available at: https://doi.org/10.26036/CNP0005902.
 
Informed consent
 
All animal procedures were approved by the Animal Welfare and Ethics Committee of Qinghai Academy of Animal Science and Veterinary Medicine.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Beraldi, D., McRae, A.F., Gratten, J., Slate, J., Visscher, P.M. and Pemberton, J.M. (2006). Development of a linkage map and mapping of phenotypic polymorphisms in a free- living population of soay sheep (Ovis aries). Genetics. 173(3): 1521-1537. 

  2. Cao, Y.H., Xu, S.S., Shen, M., Chen, Z.H., Gao, L., Lv, F.H., Xie, X.L., Wang, X.H., Yang, H., Liu, C.B., Zhou, P., Wan, P.C., Zhang, Y.S., Yang, J.Q., Pi, W.H., Hehua, E., Berry, D.P., Barbato, M., Esmailizadeh, A. and Li, M.H. (2021). Historical introgression from wild relatives enhanced climatic adaptation and resistance to Pneumonia in sheep. Molecular Biology and Evolution. 38(3): 838-855. 

  3. Deng, J., Xie, X.L., Wang, D.F., Zhao, C., Lv, F.H., Li, X., Yang, J., Yu, J.L., Shen, M., Gao, L., Yang, J.Q., Liu, M.J., Li, W.R., Wang, Y.T., Wang, F., Li, J.Q., Hehua, Ee., Liu, Y.G., Shen, Z.Q. and Li, M.H. (2020). Paternal origins and migratory episodes of domestic sheep. Current Biology: CB. 30(20): 4085-4095.e6. 

  4. Dominik, S., Henshall, J.M. and Hayes, B.J. (2012). A single nucleotide polymorphism on chromosome 10 is highly predictive for the polled phenotype in australian merino sheep. Animal Genetics. 43(4): 468-470. 

  5. Duan, S., Jiang, X., Zhang, X., Xie, L., Sun, Z., Ma, S. and Li, J. (2016). Diethylstilbestrol regulates the expression of LGR8 in mouse gubernaculum testis cells. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research. 22: 416-421. 

  6. Duijvesteijn, N., Bolormaa, S., Daetwyler, H.D. and van der Werf, J.H.J. (2018). Genomic prediction of the polled and horned phenotypes in merino sheep. Genetics Selection Evolution. 50(1): 28. 

  7. Feng, S., Ferlin, A., Truong, A., Bathgate, R., Wade, J.D., Corbett, S., Han, S., Tannour-Louet, M., Lamb, D.J., Foresta, C. and Agoulnik, A.I. (2009). INSL3/RXFP2 signaling in testicular descent. Annals of the New York Academy of Sciences. 1160: 197-204. 

  8. Gao, Y., Xi, S., Cai, B., Wu, T., Wang, Q., Kalds, P., Huang, S., Wang, Y., Han, S., Pan, M., Yang, C., Kou, Q., Ma, B., Wang, X., Zhou, S. and Chen, Y. (2025). Sheep with partial RXFP2 knockout exhibit normal horn phenotype but unilateral cryptorchidism. Journal of Integrative Agriculture. 24(9): 3698-3702. 

  9. Guo, T., Zhao, H., Yuan, C., Huang, S., Zhou, S., Lu, Z., Niu, C., Liu, J., Zhu, S., Yue, Y., Yang, Y., Wang, X., Chen, Y. and Yang, B. (2021). Selective sweeps uncovering the genetic basis of horn and adaptability traits on fine- wool sheep in China. Frontiers in Genetics. 12. https:// doi.org/10.3389/fgene.2021.604235. 

  10. He, X., Zhou, Z., Pu, Y., Chen, X., Ma, Y. and Jiang, L. (2016). Mapping the four horned locus and testing the polled locus in three Chinese sheep breeds. Animal Genetics. 47(5): 623-627. 

  11. Hu, X., Han, B., Liu, D., Li, X., Chen, C., Xu, T., Bao, G., Su, W., Li, G., Li, R., Xiang, X., Ma, W., Zhao, K. and Tian, D. (2026). RXFP2 gene variation drives horn sexual differences and polledness in sheep. Genomics. 118(3): 111249. 

  12. Hu, X.J., Yang, J., Xie, X.L., Lv, F.H., Cao, Y.H., Li, W.R., Liu, M.J., Wang, Y.T., Li, J.Q., Liu, Y.G., Ren, Y.L., Shen, Z.Q., Wang, F., Hehua, Ee., Han, J.L. and Li, M.H. (2019). The genome landscape of tibetan sheep reveals adaptive introgression from argali and the history of early human settlements on the qinghai–tibetan plateau. Molecular Biology and Evolution. 36(2): 283-303.

  13. Johnston, S.E., Beraldi, D., McRae, A.F., Pemberton, J.M. and Slate, J. (2010). Horn type and horn length genes map to the same chromosomal region in soay sheep. Heredity. 104(2): 196-205.

  14. Johnston, S.E., Gratten, J., Berenos, C., Pilkington, J.G., Clutton- Brock, T.H., Pemberton, J.M. and Slate, J. (2013). Life history trade-offs at a single locus maintain sexually selected genetic variation. Nature. 502(7469): 93-95.

  15. Kardos, M., Luikart, G., Bunch, R., Dewey, S., Edwards, W., McWilliam, S., Stephenson, J., Allendorf, F.W., Hogg, J.T. and Kijas, J. (2015). Whole genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep. Molecular Ecology. 24(22): 5616-5632.

  16. Kijas, J.W., Lenstra, J.A., Hayes, B., Boitard, S., Porto, N.L.R., San, C.M., Servin, B., McCulloch, R., Whan, V., Gietzen, K., Paiva, S., Barendse, W., Ciani, E., Raadsma, H., McEwan, J., Dalrymple, B. and International Sheep Genomics Consortium Members. (2012). Genome-wide analysis of the world’s sheep breeds reveals high levels of historic mixture and strong recent selection. PLoS Biology. 10(2): e1001258.

  17. Kumar, G.S.N., Nagaraja, C.S., Nagaraja, R., Jayashankar, M.R. and Kumar, M.A.S. (2019). Ovine relaxin family peptide receptor 2 (RXFP2) gene polymorphism-No association with cryptorchidism. The Indian Journal of Animal Sciences. 89(3): 251-254.

  18. Li, H., Du, M., Lin, X., Cao, X., Leng, L., Campo, F.M.P., Xu, D., Hou, L., Gao, X., Zhou, J., Cheng, M., Wang, J., Zhao, Q., Chen, Y., Yang, F. and Zhao, J. (2025). Multiple cell types guided by neurocytes orchestrate horn bud initiation in dairy goats. Genetics, Selection, Evolution: GSE. 57: 34.

  19. Li, L.L., Ma, S.K., Peng, W., Fang, Y.G., Duo, H.R., Fu, H.Y. and Jia, G.X. (2021). Genetic diversity and population structure of tibetan sheep breeds determined by whole genome resequencing. Tropical Animal Health and Production. 53(1): 174.

  20. Li, X., Yang, J., Shen, M., Xie, X.L., Liu, G.J., Xu, Y.X., Lv, F.H., Yang, H., Yang, Y.L., Liu, C.B., Zhou, P., Wan, P.C., Zhang, Y.S., Gao, L., Yang, J.Q., Pi, W.H., Ren, Y.L., Shen, Z.Q., Wang, F. and Li, M.H. (2020). Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits. Nature Communications. 11(1): 2815.

  21. Li, Y., Wang, Y., Yan, M., Wu, S., Liu, M. and Khan, R. (2025). Screening and functional transformation analysis of genes related to skeletal muscle development in supplemental- fed oula sheep. Animals. 15(20): 3040.

  22. Liu, F., Li, H., Meng, Z., Zhang, S., Yang, H., Hu, Y., Ciwang, R., He, J. and Pan, Z. (2025). Functional analysis of the FGF2 gene in horn development in sheep and identification of key regulatory variants. BMC Genomics.  27(4). https:// doi.org/10.1186/s12864-025-12309-y.

  23. Luan, Y., Wu, S., Wang, M., Pu, Y., Zhao, Q., Ma, Y., Jiang, L. and He, X. (2023). Identification of critical genes for ovine horn development based on transcriptome during the embryonic period. Biology. 12(4): 591.

  24. Lühken, G., Krebs, S., Rothammer, S., Küpper, J., Mioè, B., Russ, I. and Medugorac, I. (2016). The 1.78-kb insertion in the 32-untranslated region of RXFP2 does not segregate with horn status in sheep breeds with variable horn status. Genetics Selection Evolution. 48(1): 78.

  25. Metta, M., Praneeth, D.V., Vinoo, R., Sudhakar, K. and Jagadeswararao, S. (2020). Genetic polymorphism in a selective intron of ovine myostatin gene and its putative relation with carcass traits in sheep. Agricultural Science Digest-A Research Journal. 40(4): 436-439. doi: 10.18805/ag.D-5081.

  26. Montgomery, G.W., Henry, H.M., Dodds, K.G., Beattie, A.E., Wuliji, T. and Crawford, A.M. (1996). Mapping the horns (ho) locus in sheep: A further locus controlling horn development in domestic animals. The Journal of Heredity. 87(5): 358- 363.

  27. Pan, Z., Li, S., Liu, Q., Wang, Z., Zhou, Z., Di, R., Miao, B., Hu, W., Wang, X., Hu, X., Xu, Z., Wei, D., He, X., Yuan, L., Guo, X., Liang, B., Wang, R., Li, X., Cao, X. and Li, Y. (2018). Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization. Giga Science. 7(4): giy019.

  28. Sun, W., Chang, H., Ji, D., Liao, X., Du, L., Lu, S. and Kenji, T. (2007). Analysis on genetic diversity and isolation mechanism by distance of different ecological type sheep breeds in mongolia sheep group. Journal of Genetics and Genomics. 34(11): 1001-1009.

  29. Sun, W., Jin, X., Ma, Y. and Ma, S. (2024). Elucidating the genetic regulation of reproductive prolificacy in tibetan sheep through ovarian tissue sequencing. Indian Journal of Animal Research. 58(9): 1460-1473. doi: 10.18805/IJAR.BF-1787.

  30. Sun, W., Ma, S. and Ma, Y. (2023). Single-nucleotide polymorphism scanning of bone morphogenetic protein receptor gene and its correlation with the prolificacy of plateau tibetan sheep. Indian Journal of Animal Research. 57(12): 1594- 1598. doi: 10.18805/IJAR.BF-1616.

  31. Tian, D., Zhang, Z., Huang, B., Han, B., Li, X., Zhao, K., Tian, D., Zhang, Z., Huang, B., Han, B., Li, X. and Zhao, K. (2024). Genome-wide association analyses and population verification highlight the potential genetic basis of horned morphology during polled selection in tibetan sheep. Animals. 14(15): 2152.

  32. Wang, X., Zhou, G., Li, Q., Zhao, D. and Chen, Y. (2014). Discovery of SNPs in RXFP2 related to horn types in sheep. Small Ruminant Research. 116(2): 133-136.

  33. Wang, Y., Zhang, C., Wang, N., Li, Z., Heller, R., Liu, R., Zhao, Y., Han, J., Pan, X., Zheng, Z., Dai, X., Chen, C., Dou, M., Peng, S., Chen, X., Liu, J., Li, M., Wang, K., Liu, C. and Qiu, Q. (2019). Genetic basis of ruminant headgear and rapid antler regeneration. Science. 364(6446): eaav6335.

  34. Wiedemar, N. and Drögemüller, C. (2015). A 1.8 kb insertion in the 3′UTR of RXFP2 is associated with polledness in sheep. Animal Genetics. 46(4): 457-461.

  35. Woolley, S.A., Salavati, M. and Clark, E.L. (2023). Recent advances in the genomic resources for sheep. Mammalian Genome. 34(4): 545-558.

  36. Yang, H., Chu, M., Naominggaowa, Zhang, X., Shan, M., Lu, X., Pan, Z. and He, J. (2025). Tissue-specific expression, functional analysis and polymorphism of the KRT2 gene in sheep horn. Genomics. 117(2): 110990.

  37. Yuan, C., Lu, Z., Guo, T., Yue, Y., Wang, X., Wang, T., Zhang, Y., Hou, F., Niu, C., Sun, X., Zhao, H., Zhu, S., Liu, J. and Yang, B. (2021). A global analysis of CNVs in Chinese indigenous fine-wool sheep populations using whole- genome resequencing. BMC Genomics. 22(1): 78. 

  38. Zhou, W., Zhu, L., Peng, Y., Li, X., Zhang, X., Han, Z., Li, J., Bai, X., Gao, Q., Yang, R., Jiang, T. and Liu, S. (2025). Screening of molecular markers associated with hornless traits in qira black sheep. BMC Genomics. 26(1): 458.
Published In
Indian Journal of Animal Research
Article Metrics
Metrics Icon
19
Views
0
Citations
Reviewed By
Share Article
Download Article
In this Article
    Contact us
    Follow us

    Full Research Article

    Multiplex PCR-based Detection of SNPs in RXFP2 Associated with the Oula Sheep (Tibetan Sheep, Ovis Aries) Horn Phenotype

    Y
    Yifan Li1,2,3
    0009-0004-0441-8217
    M
    Mingyi Yan1,2,3
    Y
    Yi Chu1,2,3
    C
    Cui Han1,2,3
    S
    Sen Wu1,2,3,*
    1Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining, Qinghai 810016, P.R. China.
    2Plateau Livestock Genetic Resources Protection and Innovative Utilization Key Laboratory of Qinghai Province, Xining, Qinghai 810016, P.R. China.
    3Key Laboratory of Livestock and Poultry Genetics and Breeding on the Qinghai-Tibet Plateau (Qinghai), Ministry of Agriculture and Rural Affairs, Xining, Qinghai 810016, P.R. China.

    ABSTRACT

    Background: Horns, as a significant characteristic of ruminants, play an important role in their bodily structures and functions. A comprehensive analysis of the origin and evolution of horns will deepen our understanding of the biological mechanisms driving horn development. Relaxin/Insulin-like Family Peptide Receptor 2 (RXFP2) is a candidate gene associated with the development and morphology of sheep horns and a significant selection signal related to horn traits has been detected in the RXFP2 gene on chromosome 10.

    Methods: To further explore the regulatory effects of RXFP2 on horn presence or absence in Oula sheep, an important local meat breed in Qinghai, this study employed multiplex PCR to detect the selection signal of the RXFP2 gene in the genomes of 50 horned Oula sheep (wild type) and 50 hornless Oula sheep (bred within the breed).

    Result: We identified three single nucleotide polymorphisms (SNPs) associated with the horned phenotype in Oula sheep: one synonymous mutation in exon 14 (29512087:T>C) and two non-synonymous mutations in exon 17 (29505054:C>T and 29505096:C>T). This study provides additional evidence for the association between RXFP2 coding-region variants and horn phenotype in Oula sheep and offers potential molecular markers for future breeding applications after validation in larger and independent populations.

    INTRODUCTION

    The Oula sheep, a Tibetan breed native to the Qinghai-Tibet Plateau, is valued for meat and wool. It is characterized by robust physique, rapid growth, high-quality meat and strong cold adaptability (Li et al., 2025). Adult ewes weigh over 60 kg and rams average ~83.6 kg (Fig 1), demonstrating substantial meat production potential (Li et al., 2025). Grazing on natural pastures produces high-quality, nutritious mutton favored by consumers. However, horns in both sexes negatively affect carcass and pelt quality and reduce feed efficiency, hindering modern intensive breeding. Currently, no hornless Tibetan sheep breed has been established on the Qinghai-Tibet Plateau.

    Fig 1: Representative images of Oula sheep.


           
    Horn morphology was among the earliest traits investigated in sheep domestication (Kardos et al., 2015; Wang et al., 2019) and recent genomic advances have improved our ability to study such traits (Woolley et al., 2023). SNP-based marker screening and high-throughput sequencing have facilitated the identification of candidate loci and functional genes for economically important traits in Tibetan sheep (Metta et al., 2020; Sun et al., 2023, 2024). The genetic mechanisms underlying horn presence/absence are complex (Duijvesteijn et al., 2018). Early studies hypothesized three alleles at the autosomal Ho locus regulate hornlessness  (Wang et al., 2019). The “hornless locus” was mapped to chromosome 10 (Montgomery et al., 1996) and a hybrid merino × romney population narrowed it to a 50 kb region, where a 17-marker haplotype predicts horn phenotype with ~97% accuracy (Li et al., 2020). Genome-wide association studies (GWAS) identified a significant SNP near RXFP2 on chromosome 10 linked to polledness in Merino sheep (Dominik et al., 2012; Kijas et al., 2012), with additional evidence from Tibetan sheep (Tian et al., 2024). In Soay sheep, this locus also contains determinants for horn length and circumference (Beraldi et al., 2006) and RXFP2 was confirmed as a key candidate gene (Gao et al., 2025; Johnston et al., 2010). Recent discoveries have uncovered SNPs in RXFP2 that are closely associated with various horn types in sheep, suggesting a critical role in the regulation of horn development. However, similar findings have not been replicated in other wild populations. For instance, a study screening molecular markers associated with hornless traits in Qira black sheep identified several potential genetic markers, further emphasizing the genetic complexity of horn development across different sheep breeds (Cao et al., 2021; Zhou et al., 2025). Transcriptome analysis revealed RXFP2 upregulation in horn buds and identified other candidate genes including Foxl2 and Tnn (Luan et al., 2023). Recent studies have also highlighted the involvement of neural cells in horn bud initiation (Li et al., 2025) and the regulatory role of Fgf2 in sheep horn development (Liu et al., 2025).
           
    A 1.8-kb insertion in the RXFP2 3′UTR was associated with polledness in European breeds (Johnston et al., 2013; Wiedemar and Drögemüller, 2015), but this association was not consistent across all breeds (Pan et al., 2018). GWAS of Altay sheep found no significant loci and the 1.8-kb insertion was not associated with hornlessness (Deng et al., 2020). Resequencing analysis of Hu sheep identified a signal near RXFP2 on chromosome 10 (Johnston et al., 2013). Whole-genome sequencing of Chinese sheep suggested RXFP2’s role in unique horn phenotypes (Pan et al., 2018). Global analysis of copy number variations (CNVs) in Chinese indigenous fine-wool sheep populations revealed several genomic regions associated with horn development (Yuan et al., 2021), reflecting the complex inheritance of horn traits (Hu et al., 2019).
           
    However, most previous studies have focused on genomic region discovery rather than detailed characterization of functional coding variants in RXFP2 associated with horn phenotype in indigenous breeds. Despite extensive investigation of RXFP2 in several breeds, its coding-region polymorphisms have not been systematically characterized in Oula sheep.
           
    Notably, the association between RXFP2 and horn phenotype is not universal across breeds: the 1.78-kb insertion in the 3′UTR does not segregate with horn status in breeds with variable horn status (Lühken et al., 2016) and a 1.8-kb insertion in the same region was unrelated to polledness in Altay sheep (He et al., 2016). These findings highlight the need for breed-specific investigations of RXFP2 variants. Furthermore, previous studies have mainly focused on locus discovery, whereas rapid and cost-effective molecular assays for routine breeding remain limited. Therefore, we aimed to identify coding-region SNPs associated with horn phenotype in Oula sheep and establish a multiplex PCR-based genotyping method for future marker-assisted breeding.

    MATERIALS AND METHODS

    Sample collection and DNA isolation
     
    Animals were classified by external horn phenotype: horned individuals had fully developed horns; hornless individuals had horn length <0.5 cm and no visible horn structures; intermediate or scurs were excluded. Sheep were from a purebred population maintained by the Henan County Hornless Oula Sheep Breeding Demonstration Base in Qinghai Province. By 2023, our team had established 2 core and 4 demonstration flocks, with 1,138 hornless ewes (826 core + 312 replacement) and 57 hornless rams. All animals had clear phenotypic documentation and were adult ewes at 2 years of age. Initially, 100 sheep (50 horned, 50 hornless) were selected; after quality control (QC), reliable data were obtained from 48 horned and 46 hornless individuals, with balanced phenotype groups for comparative analyses. Blood samples were collected from the jugular vein and DNA was extracted using a rapid kit (Sangon Biotech, Shanghai) following the manufacturer’s protocol. DNA quality was assessed using a MaestroNano (MaestroGEN, USA). All animal procedures were approved by the Animal Care and Use Committee of the Qinghai Academy of Animal Husbandry and Veterinary Science (approval no. 2025-QHMKY-004) and conducted between 2023 and 2025.
     
    Primer design
     
    Primers were designed based on the 18 exon sequences of the RXFP2 gene available in the GeneBank database (NCBI reference sequence: NC_056063.1) [https://www.ncbi.nlm.nih.gov/] and designed using the Prime tool (Table 1) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

    Table 1: Primers for amplifying the 18 exons of the RXFP2 gene.


     
    PCR amplification
     
    A primer pool covering 18 exons was designed and tested using a two-step PCR protocol. The first PCR mixture (25 μl) contained 2 μl DNA template (10 ng/μl), 1 μl upstream primer pool (10 μM), 1 μl downstream primer pool (10 μM) and 15 μl 2× Kapa HiFi PCR ready mix. PCR conditions: 98°C for 3 min; 8 cycles of 98°C for 30 s, 50°C for 30 s, 72°C for 30 s; then 25 cycles of 98°C for 30 s, 66°C for 30 s, 72°C for 30 s; final extension at 72°C for 5 min. Products were verified on 1% agarose gel, purified with AMPure XP beads and used for second-round PCR to generate libraries. The second PCR (30 μl) contained 2 μl DNA template, 1 μl universal P7 primer (with molecular tag, 10 μM), 1 μl universal P5 primer (10 μM) and 15 μl 2× PCR ready mix. Conditions: 98°C for 5 min; 5 cycles of 94°C for 30 s, 55°C for 20 s, 72°C for 30 s; final extension at 72°C for 5 min. Products were purified, pooled and sequenced on a HiSeq XTen (Illumina, San Diego, CA). The data have been deposited in the CNGB Sequence Archive (CNSA) with accession number CNP0005902. (DOI: 10.26036/CNP0005902; available at: https://doi.org/10.26036/CNP0005902).
     
    Data quality control and genotyping analysis
     
    QC was performed in two steps: (1) removal of junction sequences using cutadapt (V.1.2.1, https://cutadapt. readthedocs.io/en/stable/); (2) removal of bases with quality score <20 using PRINSEQ-lite (V.0.20.3, https://sourceforge.net/projects/prinseq/files/). QC-passed sequences were aligned to the reference genome using BWA (V.0.7.13-r1126, https://bio-bwa.sourceforge.net/) with default parameters. Genotype data were generated using samtools (V.0.1.18, https://samtools.sourceforge.net/), gene annotation using annovar (https://annovar. openbioinformatics.org/en/latest/; NCBI reference: NC_056063.1) and haplotype analysis using SHEsis (https://github.com/celaoforever/SHEsisPlus).

    RESULTS AND DISCUSSION

    Validation and mutation analysis of coding-region SNPs in the RXFP2 using multiplex PCR
     
    Using multiplex PCR, three mutations were identified in the coding region of RXFP2 (Table 2). The first mutation, in exon 14, is a synonymous mutation (A→G, proline unchanged). The second and third mutations, both in exon 17, are non-synonymous: G→A (E→K) and G→A (V→M).

    Table 2: Mutations identified in the exonic regions of the RXFP2 gene.


           
    The three SNP loci were identified at chromosomal positions 29512087 (T>C), 29505054 (C>T) and 29505096 (C>T) (Fig 2).

    Fig 2: SNP mutation details.


     
    Genetic diversity and correlation analysis of the RXFP2 gene
     
    For the first mutation (29512087T>C), genotype frequencies were TT (21.28%), TC (29.79%) and CC (48.93%), with allele frequencies T (36.17%) and C (63.83%). Phenotype frequencies for TT, TC and CC were 100% hornless, 92.86% hornless/7.14% horned and 100% horned, respectively. For the second SNP (29505054C>T), the genotype frequencies were CC (21.28%), CT (30.85%) and TT (48.87%), with allele frequencies C (36.70%) and T (63.30%). The homozygous CC genotype was exclusively associated with the hornless phenotype, while the homozygous TT genotype was exclusively associated with the horned phenotype; heterozygous CT individuals showed a predominantly hornless phenotype (89.66% hornless vs. 10.34% horned). For the third SNP (29505096C>T), genotype frequencies were CC (22.34%), CT (36.17%) and TT (41.49%), with allele frequencies C (40.43%) and T (59.57%). Again, homozygous CC was exclusively hornless, homozygous TT exclusively horned and heterozygous CT individuals were predominantly hornless (73.53% hornless vs. 26.47% horned) (Table 3).

    Table 3: Genotype distribution of SNPs in the coding region of the RXFP2 gene.


           
    Haplotype analysis using SHEsis identified three combined haplotypes (probability > 0.03). Their frequencies in horned vs. hornless sheep were 6.3% vs. 1.1%, 90.6% vs. 27.2% and 3.1% vs. 71.7%, respectively (Table 4).

    Table 4: Cardinality test for SNPs in the coding region of the RXFP2 gene.


           
    Strong linkage disequilibrium (LD) was observed among the three SNPs (positions: 29,512,087; 29,505,096; and 29,505,054), with D′ and r2 approaching 1, indicating complete linkage disequilibrium (Fig 3, Table 5).

    Fig 3: LD correlation diagram between SNPs.



    Table 5: Analysis of linkage disequilibrium between SNP loci.


           
    RXFP2    , also known as leucine-rich G protein-coupled receptor 8 (LGR8), belongs to the G protein-coupled receptor (GPCR) family and is the specific receptor for insulin-like factor 3 (INSL-3) in the body (Duan et al., 2016; Feng et al., 2009). RXFP2 is a candidate gene associated with horn type in sheep. Beyond its well-characterized role in horn development and morphology, RXFP2 also has other physiological functions. For instance, a study investigated RXFP2 polymorphisms in Indian sheep and found no association with cryptorchidism, highlighting its functional diversity beyond horn traits (Kumar et al., 2019).RXFP2 is expressed in horn soft tissue and periosteum, with expression negatively correlated with horn size (Johnston et al., 2013). Recent studies have highlighted the complex tissue-specific expression and regulatory functions of RXFP2 in horn tissue, though it may not be the sole determinant of horn traits (Yang et al., 2025). Several GWAS have reported that RXFP2 is associated with horn presence but is not directly involved in horn development (Kijas et al., 2012).
           
    Although previous GWAS and sequencing studies have identified RXFP2 as a major candidate gene for sheep horn phenotype, these approaches primarily provide genomic association rather than practical genotyping strategies. Here, we characterized coding-region SNPs in RXFP2 in Oula sheep and developed a multiplex PCR-based detection approach. Unlike previous genome-wide studies, we focused on specific coding-region variants, including two non-synonymous mutations and established a genotyping strategy for horn-associated alleles. Thus, this study translates previous genetic findings into a cost-effective and rapid genotyping tool that can be used as part of marker-assisted selection programs in Oula sheep, with the caveat that heterozygous individuals exhibit incomplete phenotypic penetrance and thus these markers should be interpreted alongside other phenotypic or genomic information. Validation in larger, independent populations is ongoing.
           
    In the preliminary stage, horned and hornless Oula sheep populations were identified by whole-genome resequencing. Previous studies have shown that the 3′ UTR insertion in RXFP2 is associated with polledness in multiple sheep breeds (Wiedemar and Drögemüller, 2015). A strong selection signal was detected in the middle and downstream regions of RXFP2, confirming it as a major candidate gene for horn presence/absence in Oula sheep. Ovine RXFP2 encodes 763 amino acids and comprises 18 exons (Wang et al., 2014). GWAS by (Li et al., 2021) identified RXFP2 as significantly associated with horned and hornless phenotypes in sheep. A subsequent selective sweep analysis of the Manhattan plot on chromosome 10 revealed that the allele frequency distribution of a non-synonymous SNP downstream of RXFP2 across multiple breeds was consistent with our findings (Li et al., 2020). Among the three high-frequency SNPs, 29512087T>C is synonymous, while the other two are non-synonymous.
           
    The two non-synonymous variants in exon 17 resulted in amino acid substitutions (p.V653M and p.E667K) in RXFP2. A recent CRISPR/Cas9 partial knockout study in sheep showed that RXFP2 expression was significantly reduced in horn buds, but horn growth and morphology were unaffected; however, the knockout sheep exhibited unilateral cryptorchidism (Gao et al., 2025). This finding supports that RXFP2 is involved in horn phenotype determination rather than directly participating in horn development and highlights the functional relevance of its coding-region variants in horn phenotype regulation. Although the functional consequences of these substitutions require further validation, changes in amino acid properties may influence protein conformation, receptor stability, or downstream signaling. Thus, these variants represent potential functional candidates for RXFP2 variation and horn phenotype in Oula sheep.
           
    Similar coding-region polymorphisms were reported in Tan sheep, where resequencing of all 18 exons of RXFP2 identified multiple exonic SNPs, including a synonymous variant (p.P375, c.1125A>G) with distinct genotypes between horned and hornless individuals (Wang et al., 2014). Although synonymous mutations do not alter the amino acid sequence, accumulating evidence shows that they regulate gene expression through mRNA stability, codon usage bias, splicing and RNA modifications such as N6-methyladenosine (m6A). Recent studies further provided evidence that a single synonymous mutation can modulate phenotypic traits via epitranscriptomic regulation involving m6A modification and mRNA structural conformation (Zhou et al., 2025). These findings highlight the functional significance of synonymous variants such as p.P375 in RXFP2. Together with our Oula sheep data, this suggests that RXFP2 coding-region variation may contribute to horn phenotype diversity across breeds. Notably, allele frequencies at p.P375 in Tan sheep differ from those at the three SNPs in our Oula population (major allele frequencies: 36.17%–63.83%), reflecting population-specific selection or genetic backgrounds. A strong selection signal at RXFP2 has also been reported in Merino sheep, with a downstream non-synonymous SNP showing distinct allele frequencies between horned and polled populations (Dominik et al., 2012;  Li et al., 2020). In Tibetan sheep, GWAS have identified multiple SNPs in RXFP2 for horn length and base circumference, with allele frequencies varying across horn phenotypes (Hu et al., 2026; Tian et al., 2024). These cross-breed comparisons highlight the breed-specific genetic architecture of horn traits and suggest that RXFP2 variants have been subject to different selection pressures across populations. Moreover, a recent targeted sequencing study demonstrated that different RXFP2 variants exhibit breed-specific effects on horn morphology and polledness, supporting that the genetic architecture of horn traits is more complex than a single causative mutation (Hu et al., 2026). In addition, although the hornless trait is a well-known dominant trait, RXFP2 heterozygotes in our Oula population did not fully exhibit the hornless phenotype, suggesting that other regulatory effects may be involved (Wang et al., 2019), especially in our Oula sheep population and selection sweeps around RXFP2 associated with various horn traits further support a polygenic model for horn phenotype regulation (Guo et al., 2021).
           
    PIC is often associated with within population genetic diversity (Sun et al., 2007), PIC values (0.25-0.5) indicated moderate polymorphism in Oula sheep, suggesting low genetic variation and selection potential. D′ and r2 values approached 1, indicating strong LD among the three SNPs. Homozygotes showed complete phenotype concordance at each mutation site, while heterozygotes predicted hornless phenotype with 73.53% to 92.86% accuracy. Haplotype analysis revealed three haplotypes with inheritance probability >3%; CTT showed the highest predictive probability for horned (90.6%), whereas TCC showed the highest for hornless (71.7%).
           
    In Oula sheep, RXFP2 was identified as a major candidate gene for horn presence/absence. Three key SNPs in coding regions were significantly associated with horn phenotype in both horned and hornless populations and genotype identification is essential for determining horn status. These findings provide useful genetic information for understanding horn phenotype variation in Oula sheep and lay the foundation for further validation of RXFP2 variants in breeding programs.

    CONCLUSION

    In this study, multiplex PCR was used to genotype coding-region variants of the RXFP2 gene in 94 Oula sheep with distinct horn phenotypes. Three SNPs were identified: one synonymous mutation in exon 14 (29512087T>C) and two non-synonymous mutations in exon 17 (29505054C>T and 29505096C>T). Homozygous genotypes at all three loci showed complete concordance with horn phenotype, whereas heterozygotes displayed predominantly hornless phenotypes with 73.53%–92.86% accuracy. Haplotype analysis revealed that the CTT haplotype was strongly associated with the horned phenotype, while TCC was associated with the hornless phenotype. Strong linkage disequilibrium was observed among the three SNPs. These findings confirm that RXFP2 coding-region variants are associated with horn phenotype in Oula sheep and provide candidate molecular markers for marker-assisted selection after validation in larger, independent populations.

    ACKNOWLEDGEMENT

    The present study was supported by the Key Research and Transformation Project of Qinghai Province (Grant No. 2024-NK-104), entitled “Cultivation of New Variety of Hornless Oula Sheep and Integration Demonstration of Efficient Production Technologies.”
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Author contributions
     
    M.Y. Yan and S. Wu conceived and designed the study. Y.F. Li performed the experiments. Y.F. Li and Y. Chu analyzed the data and wrote the manuscript. C. Han provided reagents, materials and analysis tools. All authors reviewed and approved the final manuscript.
     
    Data availability
     
    The sequencing data generated in this study have been deposited in the CNGB Sequence Archive (CNSA) of CNGBdb under accession number CNP0005902 and are publicly available at: https://doi.org/10.26036/CNP0005902.
     
    Informed consent
     
    All animal procedures were approved by the Animal Welfare and Ethics Committee of Qinghai Academy of Animal Science and Veterinary Medicine.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Beraldi, D., McRae, A.F., Gratten, J., Slate, J., Visscher, P.M. and Pemberton, J.M. (2006). Development of a linkage map and mapping of phenotypic polymorphisms in a free- living population of soay sheep (Ovis aries). Genetics. 173(3): 1521-1537. 

    2. Cao, Y.H., Xu, S.S., Shen, M., Chen, Z.H., Gao, L., Lv, F.H., Xie, X.L., Wang, X.H., Yang, H., Liu, C.B., Zhou, P., Wan, P.C., Zhang, Y.S., Yang, J.Q., Pi, W.H., Hehua, E., Berry, D.P., Barbato, M., Esmailizadeh, A. and Li, M.H. (2021). Historical introgression from wild relatives enhanced climatic adaptation and resistance to Pneumonia in sheep. Molecular Biology and Evolution. 38(3): 838-855. 

    3. Deng, J., Xie, X.L., Wang, D.F., Zhao, C., Lv, F.H., Li, X., Yang, J., Yu, J.L., Shen, M., Gao, L., Yang, J.Q., Liu, M.J., Li, W.R., Wang, Y.T., Wang, F., Li, J.Q., Hehua, Ee., Liu, Y.G., Shen, Z.Q. and Li, M.H. (2020). Paternal origins and migratory episodes of domestic sheep. Current Biology: CB. 30(20): 4085-4095.e6. 

    4. Dominik, S., Henshall, J.M. and Hayes, B.J. (2012). A single nucleotide polymorphism on chromosome 10 is highly predictive for the polled phenotype in australian merino sheep. Animal Genetics. 43(4): 468-470. 

    5. Duan, S., Jiang, X., Zhang, X., Xie, L., Sun, Z., Ma, S. and Li, J. (2016). Diethylstilbestrol regulates the expression of LGR8 in mouse gubernaculum testis cells. Medical Science Monitor: International Medical Journal of Experimental and Clinical Research. 22: 416-421. 

    6. Duijvesteijn, N., Bolormaa, S., Daetwyler, H.D. and van der Werf, J.H.J. (2018). Genomic prediction of the polled and horned phenotypes in merino sheep. Genetics Selection Evolution. 50(1): 28. 

    7. Feng, S., Ferlin, A., Truong, A., Bathgate, R., Wade, J.D., Corbett, S., Han, S., Tannour-Louet, M., Lamb, D.J., Foresta, C. and Agoulnik, A.I. (2009). INSL3/RXFP2 signaling in testicular descent. Annals of the New York Academy of Sciences. 1160: 197-204. 

    8. Gao, Y., Xi, S., Cai, B., Wu, T., Wang, Q., Kalds, P., Huang, S., Wang, Y., Han, S., Pan, M., Yang, C., Kou, Q., Ma, B., Wang, X., Zhou, S. and Chen, Y. (2025). Sheep with partial RXFP2 knockout exhibit normal horn phenotype but unilateral cryptorchidism. Journal of Integrative Agriculture. 24(9): 3698-3702. 

    9. Guo, T., Zhao, H., Yuan, C., Huang, S., Zhou, S., Lu, Z., Niu, C., Liu, J., Zhu, S., Yue, Y., Yang, Y., Wang, X., Chen, Y. and Yang, B. (2021). Selective sweeps uncovering the genetic basis of horn and adaptability traits on fine- wool sheep in China. Frontiers in Genetics. 12. https:// doi.org/10.3389/fgene.2021.604235. 

    10. He, X., Zhou, Z., Pu, Y., Chen, X., Ma, Y. and Jiang, L. (2016). Mapping the four horned locus and testing the polled locus in three Chinese sheep breeds. Animal Genetics. 47(5): 623-627. 

    11. Hu, X., Han, B., Liu, D., Li, X., Chen, C., Xu, T., Bao, G., Su, W., Li, G., Li, R., Xiang, X., Ma, W., Zhao, K. and Tian, D. (2026). RXFP2 gene variation drives horn sexual differences and polledness in sheep. Genomics. 118(3): 111249. 

    12. Hu, X.J., Yang, J., Xie, X.L., Lv, F.H., Cao, Y.H., Li, W.R., Liu, M.J., Wang, Y.T., Li, J.Q., Liu, Y.G., Ren, Y.L., Shen, Z.Q., Wang, F., Hehua, Ee., Han, J.L. and Li, M.H. (2019). The genome landscape of tibetan sheep reveals adaptive introgression from argali and the history of early human settlements on the qinghai–tibetan plateau. Molecular Biology and Evolution. 36(2): 283-303.

    13. Johnston, S.E., Beraldi, D., McRae, A.F., Pemberton, J.M. and Slate, J. (2010). Horn type and horn length genes map to the same chromosomal region in soay sheep. Heredity. 104(2): 196-205.

    14. Johnston, S.E., Gratten, J., Berenos, C., Pilkington, J.G., Clutton- Brock, T.H., Pemberton, J.M. and Slate, J. (2013). Life history trade-offs at a single locus maintain sexually selected genetic variation. Nature. 502(7469): 93-95.

    15. Kardos, M., Luikart, G., Bunch, R., Dewey, S., Edwards, W., McWilliam, S., Stephenson, J., Allendorf, F.W., Hogg, J.T. and Kijas, J. (2015). Whole genome resequencing uncovers molecular signatures of natural and sexual selection in wild bighorn sheep. Molecular Ecology. 24(22): 5616-5632.

    16. Kijas, J.W., Lenstra, J.A., Hayes, B., Boitard, S., Porto, N.L.R., San, C.M., Servin, B., McCulloch, R., Whan, V., Gietzen, K., Paiva, S., Barendse, W., Ciani, E., Raadsma, H., McEwan, J., Dalrymple, B. and International Sheep Genomics Consortium Members. (2012). Genome-wide analysis of the world’s sheep breeds reveals high levels of historic mixture and strong recent selection. PLoS Biology. 10(2): e1001258.

    17. Kumar, G.S.N., Nagaraja, C.S., Nagaraja, R., Jayashankar, M.R. and Kumar, M.A.S. (2019). Ovine relaxin family peptide receptor 2 (RXFP2) gene polymorphism-No association with cryptorchidism. The Indian Journal of Animal Sciences. 89(3): 251-254.

    18. Li, H., Du, M., Lin, X., Cao, X., Leng, L., Campo, F.M.P., Xu, D., Hou, L., Gao, X., Zhou, J., Cheng, M., Wang, J., Zhao, Q., Chen, Y., Yang, F. and Zhao, J. (2025). Multiple cell types guided by neurocytes orchestrate horn bud initiation in dairy goats. Genetics, Selection, Evolution: GSE. 57: 34.

    19. Li, L.L., Ma, S.K., Peng, W., Fang, Y.G., Duo, H.R., Fu, H.Y. and Jia, G.X. (2021). Genetic diversity and population structure of tibetan sheep breeds determined by whole genome resequencing. Tropical Animal Health and Production. 53(1): 174.

    20. Li, X., Yang, J., Shen, M., Xie, X.L., Liu, G.J., Xu, Y.X., Lv, F.H., Yang, H., Yang, Y.L., Liu, C.B., Zhou, P., Wan, P.C., Zhang, Y.S., Gao, L., Yang, J.Q., Pi, W.H., Ren, Y.L., Shen, Z.Q., Wang, F. and Li, M.H. (2020). Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits. Nature Communications. 11(1): 2815.

    21. Li, Y., Wang, Y., Yan, M., Wu, S., Liu, M. and Khan, R. (2025). Screening and functional transformation analysis of genes related to skeletal muscle development in supplemental- fed oula sheep. Animals. 15(20): 3040.

    22. Liu, F., Li, H., Meng, Z., Zhang, S., Yang, H., Hu, Y., Ciwang, R., He, J. and Pan, Z. (2025). Functional analysis of the FGF2 gene in horn development in sheep and identification of key regulatory variants. BMC Genomics.  27(4). https:// doi.org/10.1186/s12864-025-12309-y.

    23. Luan, Y., Wu, S., Wang, M., Pu, Y., Zhao, Q., Ma, Y., Jiang, L. and He, X. (2023). Identification of critical genes for ovine horn development based on transcriptome during the embryonic period. Biology. 12(4): 591.

    24. Lühken, G., Krebs, S., Rothammer, S., Küpper, J., Mioè, B., Russ, I. and Medugorac, I. (2016). The 1.78-kb insertion in the 32-untranslated region of RXFP2 does not segregate with horn status in sheep breeds with variable horn status. Genetics Selection Evolution. 48(1): 78.

    25. Metta, M., Praneeth, D.V., Vinoo, R., Sudhakar, K. and Jagadeswararao, S. (2020). Genetic polymorphism in a selective intron of ovine myostatin gene and its putative relation with carcass traits in sheep. Agricultural Science Digest-A Research Journal. 40(4): 436-439. doi: 10.18805/ag.D-5081.

    26. Montgomery, G.W., Henry, H.M., Dodds, K.G., Beattie, A.E., Wuliji, T. and Crawford, A.M. (1996). Mapping the horns (ho) locus in sheep: A further locus controlling horn development in domestic animals. The Journal of Heredity. 87(5): 358- 363.

    27. Pan, Z., Li, S., Liu, Q., Wang, Z., Zhou, Z., Di, R., Miao, B., Hu, W., Wang, X., Hu, X., Xu, Z., Wei, D., He, X., Yuan, L., Guo, X., Liang, B., Wang, R., Li, X., Cao, X. and Li, Y. (2018). Whole-genome sequences of 89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization. Giga Science. 7(4): giy019.

    28. Sun, W., Chang, H., Ji, D., Liao, X., Du, L., Lu, S. and Kenji, T. (2007). Analysis on genetic diversity and isolation mechanism by distance of different ecological type sheep breeds in mongolia sheep group. Journal of Genetics and Genomics. 34(11): 1001-1009.

    29. Sun, W., Jin, X., Ma, Y. and Ma, S. (2024). Elucidating the genetic regulation of reproductive prolificacy in tibetan sheep through ovarian tissue sequencing. Indian Journal of Animal Research. 58(9): 1460-1473. doi: 10.18805/IJAR.BF-1787.

    30. Sun, W., Ma, S. and Ma, Y. (2023). Single-nucleotide polymorphism scanning of bone morphogenetic protein receptor gene and its correlation with the prolificacy of plateau tibetan sheep. Indian Journal of Animal Research. 57(12): 1594- 1598. doi: 10.18805/IJAR.BF-1616.

    31. Tian, D., Zhang, Z., Huang, B., Han, B., Li, X., Zhao, K., Tian, D., Zhang, Z., Huang, B., Han, B., Li, X. and Zhao, K. (2024). Genome-wide association analyses and population verification highlight the potential genetic basis of horned morphology during polled selection in tibetan sheep. Animals. 14(15): 2152.

    32. Wang, X., Zhou, G., Li, Q., Zhao, D. and Chen, Y. (2014). Discovery of SNPs in RXFP2 related to horn types in sheep. Small Ruminant Research. 116(2): 133-136.

    33. Wang, Y., Zhang, C., Wang, N., Li, Z., Heller, R., Liu, R., Zhao, Y., Han, J., Pan, X., Zheng, Z., Dai, X., Chen, C., Dou, M., Peng, S., Chen, X., Liu, J., Li, M., Wang, K., Liu, C. and Qiu, Q. (2019). Genetic basis of ruminant headgear and rapid antler regeneration. Science. 364(6446): eaav6335.

    34. Wiedemar, N. and Drögemüller, C. (2015). A 1.8 kb insertion in the 3′UTR of RXFP2 is associated with polledness in sheep. Animal Genetics. 46(4): 457-461.

    35. Woolley, S.A., Salavati, M. and Clark, E.L. (2023). Recent advances in the genomic resources for sheep. Mammalian Genome. 34(4): 545-558.

    36. Yang, H., Chu, M., Naominggaowa, Zhang, X., Shan, M., Lu, X., Pan, Z. and He, J. (2025). Tissue-specific expression, functional analysis and polymorphism of the KRT2 gene in sheep horn. Genomics. 117(2): 110990.

    37. Yuan, C., Lu, Z., Guo, T., Yue, Y., Wang, X., Wang, T., Zhang, Y., Hou, F., Niu, C., Sun, X., Zhao, H., Zhu, S., Liu, J. and Yang, B. (2021). A global analysis of CNVs in Chinese indigenous fine-wool sheep populations using whole- genome resequencing. BMC Genomics. 22(1): 78. 

    38. Zhou, W., Zhu, L., Peng, Y., Li, X., Zhang, X., Han, Z., Li, J., Bai, X., Gao, Q., Yang, R., Jiang, T. and Liu, S. (2025). Screening of molecular markers associated with hornless traits in qira black sheep. BMC Genomics. 26(1): 458.
    In this Article
      Contact us
      Follow us
      Published In
      Indian Journal of Animal Research

      Editorial Board

      View all (0)