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Full Research Article

Establishment of A PCR-SSCP-based Method for Detecting FECB Gene SNP in Tibetan Sheep

T
Taichun Li1,2,3
W
Wu Sun1,2,3,*
X
Xiayang Jin1,2,3
S
Shike Ma1,2,3
Y
Yuhong Ma1,2,3
1Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, China.
2Key Laboratory of Livestock and Poultry Genetics and Breeding on the Qinghai-Xizang Plateau, Ministry of Agriculture and Rural Affairs, Xining 810016, China.
3Key Laboratory of Genetic Resource Conservation and Innovative Utilization of Plateau Livestock in Qinghai Province, Xining 810016, China.

ABSTRACT

Background: The reproductive performance of highland Tibetan sheep is generally low, which limits the economic returns of sheep farming. As a key candidate gene influencing multiple lambing in sheep, the c.746A>G mutation in the FecB gene can significantly increase ovulation rates and litter sizes. Therefore, this study aims to optimise PCR-SSCP technology and establish a method for SNP genotyping of the FecB gene in Tibetan sheep of the Qinghai-Tibet Plateau type, thereby providing technical support and a theoretical basis for molecular marker-assisted selection and breeding aimed at high-productivity in Tibetan sheep.

Methods: Blood samples were collected from 50 healthy female Tibetan sheep from the Qinghai-Tibet Plateau and genomic DNA was extracted. Specific primers were designed for the c.746A>G mutation site in exon 8 of the FecB gene. Following PCR amplification, genotyping was performed using SSCP analysis and allele frequencies were calculated using the allele counting method to further analyse the association between different genotypes and the number of lambs born.

Result: A PCR-SSCP assay for detecting SNPs in the FecB gene of Tibetan sheep was successfully established. Two genotypes, ++ and B+, were identified, with allele frequencies of 0.67 and 0.33, respectively. Association analysis revealed that the average number of lambs born per ewe (1.82±0.21) was significantly higher in individuals with the B+ genotype than in those with the ++ genotype (1.45±0.18) (P<0.05).

INTRODUCTION

The Plateau Tibetan sheep, a distinctive livestock resource indigenous to the Qinghai-Tibet Plateau, is remarkably adapted to thrive in high-altitude environments (Li et al., 2025). This breed exhibits distinct biological traits, including exceptional environmental hardiness, high genetic stability and pronounced disease resistance (Tian et al., 2025). Its meat is tender and nutritious, making it not only an important source of mutton products but also one of the main sources of income for local herders. Due to long-term adaptation to the harsh climatic conditions of the plateau, Tibetan sheep generally exhibit low reproductive performance. This is primarily characterized by a low lambing rate and a prolonged production cycle. Typically, a ewe produces only one lamb per year, with the occurrence of multiple births per litter being relatively rare (Han et al., 2024).
       
The FecB gene affecting sheep fertility is the first major gene for multiple births discovered in sheep. The FecB gene has been identified as a missense mutation (A746G) in the bone morphogenetic protein receptor type 1B (BMPR1B) gene, located on chromosome 6 (OAR6) (Pourali Dogaheh et al., 2020). This mutation is located in exon 8 of BMPR1B, specifically designated as c.746A>G (reference sequence NM_001009784.2), resulting in a glutamine to arginine substitution at position 249 (p.Q249R) in the amino acid sequence (Zhang et al., 2020). This mutation mainly increases ovulation rate and lamb number (Pan et al., 2025; Darissa et al., 2021; Maitra et al., 2016). The effect mainly depends on the dosage, Ewes carrying one copy of the FecB mutation typically exhibit a 1.5-fold increase in ovulation rate and a 1.0-fold increase in litter size. In contrast, ewes homozygous for the mutation (carrying two copies) demonstrate a more pronounced effect, with ovulation rate and litter size increased approximately 3.0-fold and 1.5-fold (Tang et al., 2018; Yang et al., 2023). These characteristics underscore the significant potential of the FecB mutation for application in flock expansion and enhancing economic returns in sheep production.
       
The rapid development of gene mutation and structural analysis techniques, especially following the advent of polymerase chain reaction (PCR), has led to the widespread use of various PCR-based methods, continuously expanding the depth and breadth of genetic research. Single-Strand Conformation Polymorphism (SSCP) analysis is a method that detects single nucleotide variations by resolving differences in the electrophoretic mobility of single-stranded DNA molecules in a non-denaturing gel (Vignal et al., 2002). Its underlying principle is that a single nucleotide alteration (such as a transversion, insertion, deletion, or transition) alters the secondary structure conformation of single-stranded DNA, consequently changing its migration rate (Badi et al., 2021). The integration of PCR with SSCP, resulting in the PCR-SSCP technique, combines the advantages of both methods and offers benefits such as operational simplicity, rapid analysis and low cost (Kakavas, 2021). As such, it remains a commonly used method in modern genetics for detecting point mutations and short insertions or deletions.

In recent years, various methods have been used to detect polymorphism in genes. Zamani et al. (2015) studied the polymorphism of exon 2 of the BMP15 gene in Mehraban and Lori ewes through PCR-SSCP and DNA sequencing methods. Basrin et al. (2023) used PCR-RFLP to genotype BMPR1B polymorphisms. Similarly, Mishra et al. (2018) employed PCR-RFLP technique for FecB polymorphism analysis in Kajali sheep of India. Xi et al. (2022) analyzed exon 15 variations of the EPAS1 gene in Tibetan and Hu sheep using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Zhou et al. (2022) genotyped genomic DNA samples from brown ewes using PCR-based Sanger sequencing, TaqMan probes and fluorescent qPCR.
       
Compared with PCR-SSCP, PCR-RFLP relies on restriction endonuclease digestion, a process that is typically cumbersome, time-consuming and relatively expensive (Periasamy et al., 2025). DNA sequencing is costly and has a long turnaround time. The SNaPshot technique requires the use of sequencing instruments to analyze results, making it unsuitable for on-site testing; TaqMan probe detection requires a large amount of sample DNA and the probes are expensive (Liu et al., 2020). Therefore, PCR-SSCP is suitable for rapid and accurate genotyping of the FecB gene in conventional molecular laboratories.
       
In summary, this experiment used the PCR-SSCP technique with the aim of establishing a rapid detection method for SNPs in the FecB gene of plateau-type Tibetan sheep. This method helps efficiently and economically screen individuals carrying favorable FecB alleles, thereby providing direct technical support for the genetic improvement and molecular breeding of sheep reproductive performance in the Qinghai-Tibet Plateau region and can promote the sustainable development of regional animal husbandry.

MATERIALS AND METHODS

Study area
 
The experimental animals were maintained at the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park in Haibei Prefecture, Qinghai Province during the period from October 2025 to March 2026. This site is situated at 36.92°N, 100.95°E, with an average altitude exceeding 3,000 meters above sea level, representing a significant pastoral region on the Qinghai-Tibet Plateau. The terrain is predominantly mountainous plateau, covered mainly by alpine meadow vegetation. The climate is characterized as a typical plateau continental type, featuring cold, arid conditions, abundant sunshine, significant diurnal temperature variation and a short frost-free period. The experimental procedures were conducted in the laboratory of the School of Animal Science and Veterinary Medicine at Qinghai University in Qinghai Province.
 
Experimental animals
 
This experiment focused on Qinghai Tibetan sheep, selecting 50 healthy adult ewes from the naturally grazed flocks in the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park in Haibei Prefecture, Qinghai Province. The selected ewes ranged from 3 to 6 years of age (mean±SD: 4.5±1.1 years), with parity numbers ranging from 2 to 4 (mean± SD: 2.8±0.6).
 
Sample collection
 
Approximately 5 mL of whole blood was collected from the jugular vein of each animal into vacuum tubes containing the anticoagulant EDTA (Roy et al., 2024). Following collection, all blood samples were immediately placed on ice and transported to the laboratory under low-temperature conditions. For long-term storage, samples were frozen at -20°C until subsequent genomic DNA extraction.
 
Materials
 
The major reagents required for this experiment and their sources are listed in Table 1. Briefly, PBS buffer, DNA extraction solution, proteinase K, Tris-saturated phenol, 10% SDS, chloroform, isoamyl alcohol, absolute ethanol, 70% ethanol, sodium acetate (NaAc), TE buffer, DEPC water, 10× loading buffer and agarose were purchased from Beijing Solarbio Science and Technology Co., Ltd. The 2× Es Taq Master Mix (Dye), Goldview nucleic acid dye, Boric acid and acrylamide were obtained from Sangon Biotech (Shanghai) Co., Ltd.

Table 1: Major experimental reagents.


 
Instruments
 
The major instruments and equipment used in this study are listed in Table 2.

Table 2: Major instruments and equipment.


 
Extraction of genomic DNA from blood
 
Genomic DNA was extracted from thawed blood samples using the phenol-chloroform-isoamyl alcohol method. The detailed procedure was as follows: A 100 µL aliquot of blood was treated with SDS (final concentration 0.2%) and proteinase K (final concentration 100 µg/mL), followed by incubation at 60°C for 1 h for complete lysis. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the lysate, which was mixed thoroughly by gentle inversion and centrifuged at 10,000 × g for 3 min. The upper aqueous phase was carefully collected and an equal volume of chloroform:isoamyl alcohol (24:1) was added for re-extraction, followed by centrifugation at 10,000 × g for 5 min. The resulting aqueous phase was transferred to a new tube and DNA was precipitated by adding 1/10 volume of 3 mol/L sodium acetate (NaAc) and 2 volumes of ice-cold absolute ethanol with gentle mixing. The sample was then centrifuged at 10,000 × g for 10 min to pellet the DNA. The supernatant was discarded and the DNA pellet was washed with 1 mL of 70% ethanol, followed by centrifugation at 10,000 × g for 5 min. This washing step was repeated once. The pellet was air-dried in a laminar flow cabinet and finally resuspended in 30 µL of TE buffer. The extracted DNA was stored at -20°C for subsequent use.
       
The concentration and purity (A260/A280 ratio : 1.8~2.0) of the genomic DNA were measured using a spectrophotometer (Li et al., 2023). Integrity was verified by 2% agarose gel electrophoresis: DNA samples were mixed with 6× loading buffer, loaded into the wells and electrophoresed at a constant voltage of 120 V for 40 min (Lee et al., 2012). The gel was visualized under a gel imaging system. DNA samples meeting quality standards were processed for subsequent PCR amplification.
 
Design of primers
 
Primers were designed to target the c.746A>G mutation site in exon 8 of the FecB gene (reference sequence NM_001009784.2). The amplified fragment encompasses this SNP locus for subsequent SSCP analysis. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences are detailed in Table 3.

Table 3: PCR primers for the FecB gene.


 
PCR amplification of the FecB gene
 
The PCR reaction system (25 µL) and amplification conditions are detailed in Table 4 and 5, respectively. Following amplification, 5 µL of each PCR product was verified by electrophoresis on a 2% agarose gel. A DNA marker was loaded alongside the samples for reference and electrophoresis was carried out at a constant voltage of 120 V for 20 minutes (Green et al., 2019). The gel was subsequently visualized and documented under ultraviolet light using a gel imaging system. PCR products that showed a single, bright band of the expected size (252 bp) were selected for the subsequent SSCP analysis.

Table 4: PCR reaction mixture.



Table 5: PCR amplification conditions.


 
PCR-SSCP genotyping of the FecB gene
 
Mix 5 µL of non-denaturing loading buffer with 5 µL of PCR amplification product in a centrifuge tube. The mixture was heat-denatured at 98°C for 10 min, immediately transferred to ice and rapidly cooled for 10 min to facilitate the formation and stabilization of single-stranded DNA secondary structures.
       
The non-denaturing polyacrylamide gel was prepared as follows (Chory et al., 1994): Acrylamide and bis-acrylamide were combined at a 29:1 ratio and dissolved in an appropriate volume of 1× TBE buffer to achieve a final gel concentration of 14%. Ammonium persulfate and TEMED were subsequently added sequentially and the solution was mixed thoroughly and promptly cast into a gel cassette. A comb was inserted and the gel was allowed to polymerize completely, forming a three-dimensional network with a specific pore size.
       
The denatured samples were loaded into the wells of the polymerized gel. Electrophoresis was performed in 1× TBE running buffer at a constant voltage of 100 V for 5 hours in a 4°C cold room. Following electrophoresis, the gel was subjected to silver staining. The banding patterns were finally visualized and documented using a gel imaging system and genotyping was performed based on differences in band migration patterns.
 
Sanger sequencing validation
 
To confirm the accuracy of PCR-SSCP genotyping and validate the identification of the c.746A>G mutation, representative DNA samples from each genotype (++ and B+) were subjected to direct Sanger sequencing. The sequencing was performed using the same PCR primers employed for SSCP analysis (Table 3) and the reactions were completed by Sangon Biotech Co., Ltd. (Shanghai, China). The resulting sequencing chromatograms were aligned with the reference sequence (NM_001009784.2) to confirm the specific nucleotide at position c.746 and determine the mutation status of each sample.
 
Association analysis of the FecB gene with litter size
 
We analyzed litter size differences between genotypes ( ++ and B+) using one-way ANOVA with statistical significance set at P<0.05. Since only two genotype groups were compared, no post-hoc testing was required when significant differences were detected. All statistical analyses were performed using R software. The following statistical model was used:
Yij = μ + Gi + eij
 
Where,
Yij= Litter size of the jth ewe within the ith genotype.
μ= Overall mean.
Gi= Fixed effect of genotype.
eij= Random residual error.

RESULTS AND DISCUSSION

Quality assessment of genomic DNA extracted from blood samples
 
The quality of the extracted genomic DNA was assessed by 2% agarose gel electrophoresis and spectrophotometry. As shown in Fig 1, all samples exhibited sharp, discrete bands without detectable smearing, indicating good genomic integrity with no evidence of RNA contamination or degradation. Spectrophotometric analysis revealed A260/A280 ratios between 1.8 and 2.0, confirming high DNA purity suitable for subsequent experiments.

Fig 1: Agarose gel electrophoresis of extracted genomic DNA.


 
Results of PCR amplification
 
PCR amplification of blood-derived DNA using the designed primers successfully generated the target fragment of the FecB gene. As shown in Fig 2, analysis by 2% agarose gel electrophoresis revealed discrete, specific bands of the expected size (252 bp) without detectable smearing or non-specific amplification products, demonstrating their suitability for subsequent SSCP analysis.

Fig 2: Electropherogram of PCR amplification product.



SSCP analysis of PCR products
 
PCR products were subjected to SSCP genotyping using 14% non-denaturing polyacrylamide gel electrophoresis. As shown in Fig 3, two distinct banding patterns were clearly differentiated. Based on the SSCP banding profiles, these were identified as the wild-type (++) and mutant-type (B+) patterns. The wild-type pattern displayed two bands, while the mutant-type pattern consistently exhibited three bands, corresponding to the heterozygous genotype at the FecB locus.

Fig 3: SSCP analysis of FecB gene PCR products.


       
The PCR-SSCP technique has become a widely adopted method for detecting point mutations in the ovine FecB gene, owing to its cost-effectiveness and procedural simplicity. By optimizing the SSCP conditions, Chu et al. (2011) successfully distinguished the three genotypes (++, B+ and BB) in Small Tail Han sheep, with the B+ genotype displaying a characteristic three-band pattern. La et al. (2020) studied Hu sheep, Tibetan sheep, Mongolian sheep, Altay sheep and Duolang sheep. The results showed that the Hu sheep population had three genotypes: BB, B+ and ++; the Tibetan sheep, Mongolian sheep and Altay sheep populations had only two genotypes: B+ and ++; the Duolang sheep population had only one genotype: ++.
       
In this study, the PCR-SSCP technique was effectively applied to Plateau Tibetan sheep. The observed genotype patterns for ++ (two bands) and B+ (three bands) were highly consistent with those documented in previous studies, thereby confirming the robustness and general applicability of the method. Notably, the BB homozygous genotype was not detected in our study, contrasting with some other reports. This absence may be associated with the limited sample size (n=50), their single geographical source and the relatively low frequency of the B allele within this specific high-altitude sheep population.
 
Calculation of gene frequency
 
According to the SSCP genotyping results, two alleles of the FecB gene were identified in the plateau-type Tibetan sheep population, namely the wild type (+) and the mutant type (B). Two genotypes were detected: wild-type homozygotes (++) and heterozygotes (B+), while no mutant-type homozygotes (BB) were detected. Allele frequencies were calculated using the allele counting method, with the formulas as follows: P(B) = P(BB) + ½ P(B+); P(+) = P(++) + ½ P(B+). Here, P(B) and P(+) represent the frequencies of the mutant and wild-type alleles, respectively; P(BB) and P(++) represent the frequencies of their corresponding homozygotes; P(B+) represents the frequency of heterozygotes. In this study, since P(BB) = 0, the mutant allele frequency was calculated as P(B) = 0.33 and the wild-type allele frequency as P(+) = 0.67, with the results summarized in Table 6. The low frequency of the mutant allele is likely due to natural selection in the harsh plateau environment favoring survival traits over high fecundity, combined with limited population size and a lack of artificial selection. These factors have collectively restricted the increase and expansion of the FecB mutant allele.

Table 6: Genotype frequencies of the FecB gene in plateau-type Tibetan sheep.


 
Sanger sequencing validation
 
Sanger sequencing of representative samples confirmed the accuracy of the PCR-SSCP genotyping results. The sequencing chromatograms clearly demonstrated the c.746A>G polymorphism in the FecB gene: the wild-type (++) genotype displayed a single adenine (A) nucleotide peak at position c.746 (Fig 4), while the B+ genotype showed a guanine (G) nucleotide peak at the same position (Fig 5). These sequencing results directly confirmed the c.746A>G substitution, providing definitive molecular evidence validating the reliability and accuracy of the PCR-SSCP method for identifying FecB gene polymorphisms in Tibetan sheep.

Fig 4: Sanger sequencing chromatogram of the wild-type (++) genotype at the c.746 position of the FecB gene.



Fig 5: Sanger sequencing chromatogram of the B+ genotype at the c.746 position of the FecB gene.


 
Association analysis of FecB genotype with litter size
 
Chen et al. (2023) found that the average lambing number of BB genotype ewes in the first, second and third generations of Dukhan crossbreeding was significantly (P<0.05) higher than that of B+ genotype ewes in the corresponding crossbred generations and the average lambing number of ewes showed an increasing trend with the rising frequency of the BB genotype in the population. Tao et al. (2020) found that ewes with the BB genotype of the FecB gene had a higher average litter size than those with the ++ genotype (P<0.01), significantly improving the lambing performance of Luzhong meat sheep.
       
This study yielded results similar to those of previous research. The association analysis between genotype and litter size (Table 7) revealed that Tibetan sheep carrying the heterozygous FecB mutation (B+) exhibited a higher mean litter size (1.82±0.21) compared to wild-type (++) individuals (1.45±0.18). The difference was statistically significant (P<0.05), indicating that the c.746A>G mutation has a significant influence on the reproductive performance of Tibetan sheep.

Table 7: Association between FecB genotype and litter size.

CONCLUSION

This study successfully established a detection method for the FecB gene SNP in plateau Tibetan sheep based on PCR-SSCP technology, accurately identifying the c.746A>G mutation site and calculating the B allele frequency as 0.33. Association analysis has confirmed that the B+ genotype significantly increases the number of lambs born to ewes. Sanger sequencing validation of representative samples confirmed the accuracy and reliability of the PCR-SSCP genotyping method. However, several limitations persist, including the limited sample size and detection of only a single mutation site. Future improvements could involve expanding the sample size and geographical scope and extending detection to other regions of the FecB gene or related reproduction genes such as BMP15 and GDF9.
       
This study does not aim to develop new technology, but rather to systematically apply the classic, economical and reliable PCR-SSCP technique to the specific genetic resource of Highland Tibetan sheep, establishing a genotyping protocol suitable for basic-level laboratories. The method produces sharp and easily interpretable bands, providing a highly practical technical support for breeding programs and rapid screening of high-yield individuals in Tibetan sheep populations on the Qinghai-Tibet Plateau. It holds significant practical value for the conservation and utilization of local genetic resources.

ACKNOWLEDGEMENT

We sincerely acknowledge the support and cooperation of the staff at the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park and the School of Animal Science and Veterinary Medicine at Qinghai University for their invaluable contributions to this research.
 
Funding
 
This work was supported by the National Modern Agricultural Industry Technology System (CARS-39-35), the Applied Basic Research Program of Qinghai Provincial Department of Science and Technology (Grant No. 2025-ZJ-719), the Qinghai Provincial Young and Middle-aged Scientific and Technological Talent Supporting Program (Grant No. 2023QHSKXRCTJ17) and Key Open Project of Livestock and Poultry Genetics and Breeding Laboratory on the Qinghai-Tibet Plateau, Ministry of Agriculture and Rural Affairs (2025-PLGB-06).
 
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.
 
Informed consent
 
All animal experimentation procedures and handling techniques were approved by the University Committee of Qinghai University.

CONFLICT OF INTEREST

The authors do not have any actual or potential conflict of interest to declare.

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    Establishment of A PCR-SSCP-based Method for Detecting FECB Gene SNP in Tibetan Sheep

    T
    Taichun Li1,2,3
    W
    Wu Sun1,2,3,*
    X
    Xiayang Jin1,2,3
    S
    Shike Ma1,2,3
    Y
    Yuhong Ma1,2,3
    1Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, China.
    2Key Laboratory of Livestock and Poultry Genetics and Breeding on the Qinghai-Xizang Plateau, Ministry of Agriculture and Rural Affairs, Xining 810016, China.
    3Key Laboratory of Genetic Resource Conservation and Innovative Utilization of Plateau Livestock in Qinghai Province, Xining 810016, China.

    ABSTRACT

    Background: The reproductive performance of highland Tibetan sheep is generally low, which limits the economic returns of sheep farming. As a key candidate gene influencing multiple lambing in sheep, the c.746A>G mutation in the FecB gene can significantly increase ovulation rates and litter sizes. Therefore, this study aims to optimise PCR-SSCP technology and establish a method for SNP genotyping of the FecB gene in Tibetan sheep of the Qinghai-Tibet Plateau type, thereby providing technical support and a theoretical basis for molecular marker-assisted selection and breeding aimed at high-productivity in Tibetan sheep.

    Methods: Blood samples were collected from 50 healthy female Tibetan sheep from the Qinghai-Tibet Plateau and genomic DNA was extracted. Specific primers were designed for the c.746A>G mutation site in exon 8 of the FecB gene. Following PCR amplification, genotyping was performed using SSCP analysis and allele frequencies were calculated using the allele counting method to further analyse the association between different genotypes and the number of lambs born.

    Result: A PCR-SSCP assay for detecting SNPs in the FecB gene of Tibetan sheep was successfully established. Two genotypes, ++ and B+, were identified, with allele frequencies of 0.67 and 0.33, respectively. Association analysis revealed that the average number of lambs born per ewe (1.82±0.21) was significantly higher in individuals with the B+ genotype than in those with the ++ genotype (1.45±0.18) (P<0.05).

    INTRODUCTION

    The Plateau Tibetan sheep, a distinctive livestock resource indigenous to the Qinghai-Tibet Plateau, is remarkably adapted to thrive in high-altitude environments (Li et al., 2025). This breed exhibits distinct biological traits, including exceptional environmental hardiness, high genetic stability and pronounced disease resistance (Tian et al., 2025). Its meat is tender and nutritious, making it not only an important source of mutton products but also one of the main sources of income for local herders. Due to long-term adaptation to the harsh climatic conditions of the plateau, Tibetan sheep generally exhibit low reproductive performance. This is primarily characterized by a low lambing rate and a prolonged production cycle. Typically, a ewe produces only one lamb per year, with the occurrence of multiple births per litter being relatively rare (Han et al., 2024).
           
    The FecB gene affecting sheep fertility is the first major gene for multiple births discovered in sheep. The FecB gene has been identified as a missense mutation (A746G) in the bone morphogenetic protein receptor type 1B (BMPR1B) gene, located on chromosome 6 (OAR6) (Pourali Dogaheh et al., 2020). This mutation is located in exon 8 of BMPR1B, specifically designated as c.746A>G (reference sequence NM_001009784.2), resulting in a glutamine to arginine substitution at position 249 (p.Q249R) in the amino acid sequence (Zhang et al., 2020). This mutation mainly increases ovulation rate and lamb number (Pan et al., 2025; Darissa et al., 2021; Maitra et al., 2016). The effect mainly depends on the dosage, Ewes carrying one copy of the FecB mutation typically exhibit a 1.5-fold increase in ovulation rate and a 1.0-fold increase in litter size. In contrast, ewes homozygous for the mutation (carrying two copies) demonstrate a more pronounced effect, with ovulation rate and litter size increased approximately 3.0-fold and 1.5-fold (Tang et al., 2018; Yang et al., 2023). These characteristics underscore the significant potential of the FecB mutation for application in flock expansion and enhancing economic returns in sheep production.
           
    The rapid development of gene mutation and structural analysis techniques, especially following the advent of polymerase chain reaction (PCR), has led to the widespread use of various PCR-based methods, continuously expanding the depth and breadth of genetic research. Single-Strand Conformation Polymorphism (SSCP) analysis is a method that detects single nucleotide variations by resolving differences in the electrophoretic mobility of single-stranded DNA molecules in a non-denaturing gel (Vignal et al., 2002). Its underlying principle is that a single nucleotide alteration (such as a transversion, insertion, deletion, or transition) alters the secondary structure conformation of single-stranded DNA, consequently changing its migration rate (Badi et al., 2021). The integration of PCR with SSCP, resulting in the PCR-SSCP technique, combines the advantages of both methods and offers benefits such as operational simplicity, rapid analysis and low cost (Kakavas, 2021). As such, it remains a commonly used method in modern genetics for detecting point mutations and short insertions or deletions.

    In recent years, various methods have been used to detect polymorphism in genes. Zamani et al. (2015) studied the polymorphism of exon 2 of the BMP15 gene in Mehraban and Lori ewes through PCR-SSCP and DNA sequencing methods. Basrin et al. (2023) used PCR-RFLP to genotype BMPR1B polymorphisms. Similarly, Mishra et al. (2018) employed PCR-RFLP technique for FecB polymorphism analysis in Kajali sheep of India. Xi et al. (2022) analyzed exon 15 variations of the EPAS1 gene in Tibetan and Hu sheep using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Zhou et al. (2022) genotyped genomic DNA samples from brown ewes using PCR-based Sanger sequencing, TaqMan probes and fluorescent qPCR.
           
    Compared with PCR-SSCP, PCR-RFLP relies on restriction endonuclease digestion, a process that is typically cumbersome, time-consuming and relatively expensive (Periasamy et al., 2025). DNA sequencing is costly and has a long turnaround time. The SNaPshot technique requires the use of sequencing instruments to analyze results, making it unsuitable for on-site testing; TaqMan probe detection requires a large amount of sample DNA and the probes are expensive (Liu et al., 2020). Therefore, PCR-SSCP is suitable for rapid and accurate genotyping of the FecB gene in conventional molecular laboratories.
           
    In summary, this experiment used the PCR-SSCP technique with the aim of establishing a rapid detection method for SNPs in the FecB gene of plateau-type Tibetan sheep. This method helps efficiently and economically screen individuals carrying favorable FecB alleles, thereby providing direct technical support for the genetic improvement and molecular breeding of sheep reproductive performance in the Qinghai-Tibet Plateau region and can promote the sustainable development of regional animal husbandry.

    MATERIALS AND METHODS

    Study area
     
    The experimental animals were maintained at the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park in Haibei Prefecture, Qinghai Province during the period from October 2025 to March 2026. This site is situated at 36.92°N, 100.95°E, with an average altitude exceeding 3,000 meters above sea level, representing a significant pastoral region on the Qinghai-Tibet Plateau. The terrain is predominantly mountainous plateau, covered mainly by alpine meadow vegetation. The climate is characterized as a typical plateau continental type, featuring cold, arid conditions, abundant sunshine, significant diurnal temperature variation and a short frost-free period. The experimental procedures were conducted in the laboratory of the School of Animal Science and Veterinary Medicine at Qinghai University in Qinghai Province.
     
    Experimental animals
     
    This experiment focused on Qinghai Tibetan sheep, selecting 50 healthy adult ewes from the naturally grazed flocks in the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park in Haibei Prefecture, Qinghai Province. The selected ewes ranged from 3 to 6 years of age (mean±SD: 4.5±1.1 years), with parity numbers ranging from 2 to 4 (mean± SD: 2.8±0.6).
     
    Sample collection
     
    Approximately 5 mL of whole blood was collected from the jugular vein of each animal into vacuum tubes containing the anticoagulant EDTA (Roy et al., 2024). Following collection, all blood samples were immediately placed on ice and transported to the laboratory under low-temperature conditions. For long-term storage, samples were frozen at -20°C until subsequent genomic DNA extraction.
     
    Materials
     
    The major reagents required for this experiment and their sources are listed in Table 1. Briefly, PBS buffer, DNA extraction solution, proteinase K, Tris-saturated phenol, 10% SDS, chloroform, isoamyl alcohol, absolute ethanol, 70% ethanol, sodium acetate (NaAc), TE buffer, DEPC water, 10× loading buffer and agarose were purchased from Beijing Solarbio Science and Technology Co., Ltd. The 2× Es Taq Master Mix (Dye), Goldview nucleic acid dye, Boric acid and acrylamide were obtained from Sangon Biotech (Shanghai) Co., Ltd.

    Table 1: Major experimental reagents.


     
    Instruments
     
    The major instruments and equipment used in this study are listed in Table 2.

    Table 2: Major instruments and equipment.


     
    Extraction of genomic DNA from blood
     
    Genomic DNA was extracted from thawed blood samples using the phenol-chloroform-isoamyl alcohol method. The detailed procedure was as follows: A 100 µL aliquot of blood was treated with SDS (final concentration 0.2%) and proteinase K (final concentration 100 µg/mL), followed by incubation at 60°C for 1 h for complete lysis. An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the lysate, which was mixed thoroughly by gentle inversion and centrifuged at 10,000 × g for 3 min. The upper aqueous phase was carefully collected and an equal volume of chloroform:isoamyl alcohol (24:1) was added for re-extraction, followed by centrifugation at 10,000 × g for 5 min. The resulting aqueous phase was transferred to a new tube and DNA was precipitated by adding 1/10 volume of 3 mol/L sodium acetate (NaAc) and 2 volumes of ice-cold absolute ethanol with gentle mixing. The sample was then centrifuged at 10,000 × g for 10 min to pellet the DNA. The supernatant was discarded and the DNA pellet was washed with 1 mL of 70% ethanol, followed by centrifugation at 10,000 × g for 5 min. This washing step was repeated once. The pellet was air-dried in a laminar flow cabinet and finally resuspended in 30 µL of TE buffer. The extracted DNA was stored at -20°C for subsequent use.
           
    The concentration and purity (A260/A280 ratio : 1.8~2.0) of the genomic DNA were measured using a spectrophotometer (Li et al., 2023). Integrity was verified by 2% agarose gel electrophoresis: DNA samples were mixed with 6× loading buffer, loaded into the wells and electrophoresed at a constant voltage of 120 V for 40 min (Lee et al., 2012). The gel was visualized under a gel imaging system. DNA samples meeting quality standards were processed for subsequent PCR amplification.
     
    Design of primers
     
    Primers were designed to target the c.746A>G mutation site in exon 8 of the FecB gene (reference sequence NM_001009784.2). The amplified fragment encompasses this SNP locus for subsequent SSCP analysis. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences are detailed in Table 3.

    Table 3: PCR primers for the FecB gene.


     
    PCR amplification of the FecB gene
     
    The PCR reaction system (25 µL) and amplification conditions are detailed in Table 4 and 5, respectively. Following amplification, 5 µL of each PCR product was verified by electrophoresis on a 2% agarose gel. A DNA marker was loaded alongside the samples for reference and electrophoresis was carried out at a constant voltage of 120 V for 20 minutes (Green et al., 2019). The gel was subsequently visualized and documented under ultraviolet light using a gel imaging system. PCR products that showed a single, bright band of the expected size (252 bp) were selected for the subsequent SSCP analysis.

    Table 4: PCR reaction mixture.



    Table 5: PCR amplification conditions.


     
    PCR-SSCP genotyping of the FecB gene
     
    Mix 5 µL of non-denaturing loading buffer with 5 µL of PCR amplification product in a centrifuge tube. The mixture was heat-denatured at 98°C for 10 min, immediately transferred to ice and rapidly cooled for 10 min to facilitate the formation and stabilization of single-stranded DNA secondary structures.
           
    The non-denaturing polyacrylamide gel was prepared as follows (Chory et al., 1994): Acrylamide and bis-acrylamide were combined at a 29:1 ratio and dissolved in an appropriate volume of 1× TBE buffer to achieve a final gel concentration of 14%. Ammonium persulfate and TEMED were subsequently added sequentially and the solution was mixed thoroughly and promptly cast into a gel cassette. A comb was inserted and the gel was allowed to polymerize completely, forming a three-dimensional network with a specific pore size.
           
    The denatured samples were loaded into the wells of the polymerized gel. Electrophoresis was performed in 1× TBE running buffer at a constant voltage of 100 V for 5 hours in a 4°C cold room. Following electrophoresis, the gel was subjected to silver staining. The banding patterns were finally visualized and documented using a gel imaging system and genotyping was performed based on differences in band migration patterns.
     
    Sanger sequencing validation
     
    To confirm the accuracy of PCR-SSCP genotyping and validate the identification of the c.746A>G mutation, representative DNA samples from each genotype (++ and B+) were subjected to direct Sanger sequencing. The sequencing was performed using the same PCR primers employed for SSCP analysis (Table 3) and the reactions were completed by Sangon Biotech Co., Ltd. (Shanghai, China). The resulting sequencing chromatograms were aligned with the reference sequence (NM_001009784.2) to confirm the specific nucleotide at position c.746 and determine the mutation status of each sample.
     
    Association analysis of the FecB gene with litter size
     
    We analyzed litter size differences between genotypes ( ++ and B+) using one-way ANOVA with statistical significance set at P<0.05. Since only two genotype groups were compared, no post-hoc testing was required when significant differences were detected. All statistical analyses were performed using R software. The following statistical model was used:
    Yij = μ + Gi + eij
     
    Where,
    Yij= Litter size of the jth ewe within the ith genotype.
    μ= Overall mean.
    Gi= Fixed effect of genotype.
    eij= Random residual error.

    RESULTS AND DISCUSSION

    Quality assessment of genomic DNA extracted from blood samples
     
    The quality of the extracted genomic DNA was assessed by 2% agarose gel electrophoresis and spectrophotometry. As shown in Fig 1, all samples exhibited sharp, discrete bands without detectable smearing, indicating good genomic integrity with no evidence of RNA contamination or degradation. Spectrophotometric analysis revealed A260/A280 ratios between 1.8 and 2.0, confirming high DNA purity suitable for subsequent experiments.

    Fig 1: Agarose gel electrophoresis of extracted genomic DNA.


     
    Results of PCR amplification
     
    PCR amplification of blood-derived DNA using the designed primers successfully generated the target fragment of the FecB gene. As shown in Fig 2, analysis by 2% agarose gel electrophoresis revealed discrete, specific bands of the expected size (252 bp) without detectable smearing or non-specific amplification products, demonstrating their suitability for subsequent SSCP analysis.

    Fig 2: Electropherogram of PCR amplification product.



    SSCP analysis of PCR products
     
    PCR products were subjected to SSCP genotyping using 14% non-denaturing polyacrylamide gel electrophoresis. As shown in Fig 3, two distinct banding patterns were clearly differentiated. Based on the SSCP banding profiles, these were identified as the wild-type (++) and mutant-type (B+) patterns. The wild-type pattern displayed two bands, while the mutant-type pattern consistently exhibited three bands, corresponding to the heterozygous genotype at the FecB locus.

    Fig 3: SSCP analysis of FecB gene PCR products.


           
    The PCR-SSCP technique has become a widely adopted method for detecting point mutations in the ovine FecB gene, owing to its cost-effectiveness and procedural simplicity. By optimizing the SSCP conditions, Chu et al. (2011) successfully distinguished the three genotypes (++, B+ and BB) in Small Tail Han sheep, with the B+ genotype displaying a characteristic three-band pattern. La et al. (2020) studied Hu sheep, Tibetan sheep, Mongolian sheep, Altay sheep and Duolang sheep. The results showed that the Hu sheep population had three genotypes: BB, B+ and ++; the Tibetan sheep, Mongolian sheep and Altay sheep populations had only two genotypes: B+ and ++; the Duolang sheep population had only one genotype: ++.
           
    In this study, the PCR-SSCP technique was effectively applied to Plateau Tibetan sheep. The observed genotype patterns for ++ (two bands) and B+ (three bands) were highly consistent with those documented in previous studies, thereby confirming the robustness and general applicability of the method. Notably, the BB homozygous genotype was not detected in our study, contrasting with some other reports. This absence may be associated with the limited sample size (n=50), their single geographical source and the relatively low frequency of the B allele within this specific high-altitude sheep population.
     
    Calculation of gene frequency
     
    According to the SSCP genotyping results, two alleles of the FecB gene were identified in the plateau-type Tibetan sheep population, namely the wild type (+) and the mutant type (B). Two genotypes were detected: wild-type homozygotes (++) and heterozygotes (B+), while no mutant-type homozygotes (BB) were detected. Allele frequencies were calculated using the allele counting method, with the formulas as follows: P(B) = P(BB) + ½ P(B+); P(+) = P(++) + ½ P(B+). Here, P(B) and P(+) represent the frequencies of the mutant and wild-type alleles, respectively; P(BB) and P(++) represent the frequencies of their corresponding homozygotes; P(B+) represents the frequency of heterozygotes. In this study, since P(BB) = 0, the mutant allele frequency was calculated as P(B) = 0.33 and the wild-type allele frequency as P(+) = 0.67, with the results summarized in Table 6. The low frequency of the mutant allele is likely due to natural selection in the harsh plateau environment favoring survival traits over high fecundity, combined with limited population size and a lack of artificial selection. These factors have collectively restricted the increase and expansion of the FecB mutant allele.

    Table 6: Genotype frequencies of the FecB gene in plateau-type Tibetan sheep.


     
    Sanger sequencing validation
     
    Sanger sequencing of representative samples confirmed the accuracy of the PCR-SSCP genotyping results. The sequencing chromatograms clearly demonstrated the c.746A>G polymorphism in the FecB gene: the wild-type (++) genotype displayed a single adenine (A) nucleotide peak at position c.746 (Fig 4), while the B+ genotype showed a guanine (G) nucleotide peak at the same position (Fig 5). These sequencing results directly confirmed the c.746A>G substitution, providing definitive molecular evidence validating the reliability and accuracy of the PCR-SSCP method for identifying FecB gene polymorphisms in Tibetan sheep.

    Fig 4: Sanger sequencing chromatogram of the wild-type (++) genotype at the c.746 position of the FecB gene.



    Fig 5: Sanger sequencing chromatogram of the B+ genotype at the c.746 position of the FecB gene.


     
    Association analysis of FecB genotype with litter size
     
    Chen et al. (2023) found that the average lambing number of BB genotype ewes in the first, second and third generations of Dukhan crossbreeding was significantly (P<0.05) higher than that of B+ genotype ewes in the corresponding crossbred generations and the average lambing number of ewes showed an increasing trend with the rising frequency of the BB genotype in the population. Tao et al. (2020) found that ewes with the BB genotype of the FecB gene had a higher average litter size than those with the ++ genotype (P<0.01), significantly improving the lambing performance of Luzhong meat sheep.
           
    This study yielded results similar to those of previous research. The association analysis between genotype and litter size (Table 7) revealed that Tibetan sheep carrying the heterozygous FecB mutation (B+) exhibited a higher mean litter size (1.82±0.21) compared to wild-type (++) individuals (1.45±0.18). The difference was statistically significant (P<0.05), indicating that the c.746A>G mutation has a significant influence on the reproductive performance of Tibetan sheep.

    Table 7: Association between FecB genotype and litter size.

    CONCLUSION

    This study successfully established a detection method for the FecB gene SNP in plateau Tibetan sheep based on PCR-SSCP technology, accurately identifying the c.746A>G mutation site and calculating the B allele frequency as 0.33. Association analysis has confirmed that the B+ genotype significantly increases the number of lambs born to ewes. Sanger sequencing validation of representative samples confirmed the accuracy and reliability of the PCR-SSCP genotyping method. However, several limitations persist, including the limited sample size and detection of only a single mutation site. Future improvements could involve expanding the sample size and geographical scope and extending detection to other regions of the FecB gene or related reproduction genes such as BMP15 and GDF9.
           
    This study does not aim to develop new technology, but rather to systematically apply the classic, economical and reliable PCR-SSCP technique to the specific genetic resource of Highland Tibetan sheep, establishing a genotyping protocol suitable for basic-level laboratories. The method produces sharp and easily interpretable bands, providing a highly practical technical support for breeding programs and rapid screening of high-yield individuals in Tibetan sheep populations on the Qinghai-Tibet Plateau. It holds significant practical value for the conservation and utilization of local genetic resources.

    ACKNOWLEDGEMENT

    We sincerely acknowledge the support and cooperation of the staff at the Plateau Ecological Animal Husbandry Science and Technology Demonstration Park and the School of Animal Science and Veterinary Medicine at Qinghai University for their invaluable contributions to this research.
     
    Funding
     
    This work was supported by the National Modern Agricultural Industry Technology System (CARS-39-35), the Applied Basic Research Program of Qinghai Provincial Department of Science and Technology (Grant No. 2025-ZJ-719), the Qinghai Provincial Young and Middle-aged Scientific and Technological Talent Supporting Program (Grant No. 2023QHSKXRCTJ17) and Key Open Project of Livestock and Poultry Genetics and Breeding Laboratory on the Qinghai-Tibet Plateau, Ministry of Agriculture and Rural Affairs (2025-PLGB-06).
     
    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.
     
    Informed consent
     
    All animal experimentation procedures and handling techniques were approved by the University Committee of Qinghai University.

    CONFLICT OF INTEREST

    The authors do not have any actual or potential conflict of interest to declare.

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