The plateau-type Tibetan sheep is an important indigenous sheep population distributed on the Qinghai-Tibet Plateau and is characterized by relatively low reproductive performance. Tibetan sheep generally produce one lamb per year, although individuals with higher prolificacy also occur within the population (Han et al., 2024). Compared with highly prolific breeds such as Small-tailed Han sheep and Hu sheep, plateau-type Tibetan sheep generally have a lower litter size (Han et al., 2024). Nevertheless, Tibetan sheep have undergone long-term adaptation to the hypoxic and cold environment of the Qinghai-Tibet Plateau, with genomic studies identifying multiple biological processes and candidate genes associated with adaptation to different altitudes (Zhang et al., 2024). High-altitude stress may also influence reproductive performance by affecting ovarian follicular development and reproductive hormone-related signaling pathways (Li et al., 2022). Therefore, improving reproductive performance is important for increasing production efficiency and accelerating genetic improvement in this population.
       
GDF9 (Growth Differentiation Factor 9), a member of the transforming growth factor-β (TGF-β) superfamily, is an important candidate gene involved in ovarian follicular development, ovulation and reproductive performance in sheep. Recent studies have identified multiple polymorphic loci in the GDF9 gene and evaluated their associations with litter size in different sheep populations (Wang et al., 2021; Zhang et al., 2025). For example, Wang et al. (2021) identified several GDF9 SNPs in Luzhong mutton sheep, some of which were significantly associated with litter size. More recently, genetic variation in GDF9 has been investigated across global sheep populations, further demonstrating the importance of this gene in reproductive genetics (Liu et al., 2025). Recent population-specific studies further indicate that the effects of GDF9 variants on litter size can differ among breeds and loci, emphasizing the need for breed-specific validation (Ji et al., 2023; Mura et al., 2025; KirikCI, 2023).
       
PCR-SSCP is an effective and relatively low-cost approach for screening gene polymorphisms. Recently, Hasanain et al. (2025) successfully combined PCR-SSCP and sequencing to detect GDF9 polymorphisms in sheep and investigated their associations with reproductive traits. However, information on GDF9 polymorphisms in plateau-type Tibetan sheep remains limited and a standardized PCR-SSCP system for screening GDF9 SNPs in this population has not been well established. Recent candidate-gene studies have also identified polymorphisms in GDF9, BMP15 and other fecundity-related genes and evaluated their associations with litter size in sheep, further supporting the value of targeted genetic screening for reproductive traits (Ji et al., 2023).
       
To date, no reports have been found on the development of a PCR-SSCP method specifically for detecting GDF9 gene SNPs in plateau-type Tibetan sheep. This study aimed to establish and optimize a PCR-SSCP method for detecting GDF9 gene SNPs in plateau-type Tibetan sheep, analyze the genotype and allele frequencies of the identified SNPs and preliminarily evaluate their association with litter size. The results are expected to provide a methodological basis for larger-scale validation of GDF9 polymorphisms and their potential application in the molecular breeding of plateau-type Tibetan sheep.

Sample collection
 
The study was conducted in January 2026 at the Qinghai University Academy of Animal Husbandry and Veterinary Science, Xining, Qinghai, China. Animal sampling was carried out at the Haibei Prefecture Plateau Ecological Animal Husbandry Science and Technology Demonstration Park in Qinghai Province. Fifty healthy female plateau-type Tibetan sheep were selected from the same flock at the demonstration park. Approximately 5 mL of blood was collected from the jugular vein of each animal into EDTA-containing blood collection tubes. The collected blood samples were transported to the laboratory under chilled conditions and subsequently stored at -20°C until DNA extraction. The present study was designed primarily to establish and optimize a PCR-SSCP-based method for GDF9 SNP detection and to preliminarily evaluate the association between the identified SNP and litter size. Given the exploratory nature of the study, 50 animals were included. The relatively small sample size was recognized as a limitation of the present study and further validation in larger and independent populations is required.
 
Ethics statement
 
All procedures involving animals were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of the Qinghai Academy of Animal Husbandry and Veterinary Science (Approval No. 202601457). All animal handling and blood collection procedures were conducted in accordance with the approved guidelines for animal welfare and experimental procedures.
 
Experimental reagents and instruments
 
Test reagents
 
The reagents required for this experiment include: PBS buffer, DNA extraction solution, proteinase K, anhydrous ethanol, 70% ethanol, TE buffer, DEPC-treated water, 10× Loading Buffer, agarose, etc. All the above reagents were purchased from Beijing Solarbio Science  and Technology Co., Ltd. The 2× Es Taq Master Mix (Dye), Goldview nucleic acid dye, acrylamide and Animal Genomic DNA Rapid Extraction Kit (B518221) were purchased from Sangon Biotech (Shanghai) Co., Ltd.
 
Main instruments and equipment
 
Instruments and Equipment (Table 1).


 
Blood genomic DNA extraction
 
The frozen blood samples were taken out and slowly thawed on ice. Genomic DNA was extracted according to the instructions of the Animal Genomic DNA Rapid Extraction Kit (B518221). After extraction, the concentration and purity of the extracted genomic DNA were measured using a spectrophotometer. The quality of the genomic DNA was then assessed by 2% agarose gel electrophoresis. The extracted genomic DNA was mixed with loading buffer, loaded into the wells of the prepared agarose gel and electrophoresed at 120 V for 40 min for observation. Subsequent PCR amplification was carried out only after the quality of the genomic DNA met the required standards.
 
Primer design
 
Primers were designed targeting the c.1111G>A mutation site in exon 2 of the GDF9 gene (reference sequence NM_001142888.2). The amplified fragment contains this SNP site and was intended for subsequent SSCP analysis. This locus has been reported to be significantly associated with reproductive traits in sheep. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Table 2).


 
PCR amplification
 
The GDF9 gene was amplified using the designed primers. The PCR reaction system is shown in Table 3.


       
Using the extracted whole blood DNA of plateau-type Tibetan sheep as a template, PCR amplification was performed. The PCR reaction system was 25 μL. The reagents were prepared in the order listed in the table above and mixed well (Table 4).


 
SSCP analysis
 
PCR products were subjected to SSCP analysis for SNP screening. Five microliters of PCR product were mixed with 5 μL of loading buffer and denatured at 98°C for 16 min. The denatured samples were immediately cooled at 20°C for 10 min to minimize reannealing and maintain single-stranded DNA conformations. The samples were then separated on a 14% non-denaturing polyacrylamide gel at a constant voltage of 100 V for 5 h. The gel concentration and electrophoresis settings were selected with reference to recent ovine PCR-SSCP studies. Bai et al. (2024) used 14% acrylamide/bisacrylamide gels for ovine SSCP analysis, Abhilash et al. (2022) demonstrated the continued applicability of PCR-SSCP for detecting polymorphisms in sheep. After electrophoresis, the gels were stained with silver nitrate and the resulting banding patterns were photographed and analyzed. Samples exhibiting different SSCP patterns were subsequently subjected to sequencing for SNP verification.
 
Genotype frequency and hardy-weinberg equilibrium analysis
 
Genotype and allele frequencies of the identified GDF9 SNP were calculated based on the observed genotype counts. Hardy-Weinberg equilibrium (HWE) was evaluated using an exact test, with P<0.05 considered to indicate significant deviation from HWE.
 
Association analysis of litter size
 
One-way analysis of variance (ANOVA) was used to compare the differences in litter size among different genotypes (AA/AB), with the significance threshold set at P<0.05. Data analysis was performed using R language and post-hoc tests were conducted using Tukey HSD method. The statistical model was:
 

Y_ij = μ+G_i +e_ij

 
Y_ij  = Litter size of the j^-th individual with the i-th genotype.
μ = Population mean.
G_i= The genotype effect.
e_ij = Random error.

Genomic DNA extraction results
 
After DNA extraction from the blood samples was completed, the DNA concentration was measured and the OD value was found to be within the acceptable range of 1.8-2.0. The DNA was further analyzed using 2% agarose gel electrophoresis. The electrophoresis results revealed a clear (Fig 1), single whole-genome DNA band with no additional bands observed. This indicates that the genomic DNA sample has good integrity, with no degradation or contamination. It meets the experimental requirements and can be successfully used for subsequent PCR amplification.


 
PCR amplification results
 
The product obtained by PCR amplification (Fig 2) exhibits characteristics such as consistent size with the desired target, no non-specific amplification bands and a clear image and can be used for SSCP analysis.


 
Results of PCR-SSCP polymorphism detection
 
After performing SSCP analysis on the amplified products, two different banding patterns were observed (Fig 3). Based on the analysis, these two banding patterns were defined as the AA type and the AB type. Sequencing verification revealed that AB-type individuals carry a G>A heterozygous mutation at position c.1111 in exon 2 of the GDF9 gene (Fig 4), while AA-type individuals are wild-type G/G homozygotes. This substitution results in a valine-to-methionine change at position 371 of the GDF9 protein (p.Val371Met; V371M).




 
Genotype and allele frequencies and hardy-weinberg equilibrium
 
Two alleles, A and B and two genotypes, AA and AB, were detected at the GDF9 c.1111G>A locus, whereas the BB genotype was not observed. Among the 50 plateau-type Tibetan sheep, 20 animals were classified as AA and 30 as AB, corresponding to genotype frequencies of 0.40 and 0.60, respectively. The frequencies of the A and B alleles were 0.70 and 0.30, respectively. Under Hardy-Weinberg equilibrium, the expected numbers of AA, AB and BB individuals were 24.50, 21.00 and 4.50, respectively. The exact HWE test indicated that the genotype distribution significantly deviated from Hardy-Weinberg equilibrium (P= 0.002). The absence of the BB genotype may partly reflect the relatively small sample size and the sampling of animals from a single flock; therefore, its actual frequency should be further evaluated in larger and independent populations (Table 5).


 
Association analysis of GDF9 genotype with litter size
 
The mean litter size of ewes with the AB genotype (1.85±0.18) was significantly higher than that of ewes with the AA genotype (1.52±0.15) (P= 0.035; Table 6). These results indicate a significant association between the GDF9 c.1111G>A polymorphism and litter size in the plateau-type Tibetan sheep examined in this study. However, given the relatively small sample size, this association should be considered preliminary and requires further validation in larger and independent populations.


 
Association of GDF9 gene SNP with litter size in plateau-type tibetan sheep
 
GDF9 is an important candidate gene involved in the regulation of ovarian follicular development and reproductive performance in sheep. Recent studies have demonstrated associations between GDF9 polymorphisms and reproductive traits in different sheep populations (Wang et al., 2021; Hasanain et al., 2025; Yang et al., 2026). In plateau-type Tibetan sheep, transcriptomic analysis of ovarian tissues from ewes with different litter sizes also identified GDF9, BMP15 and BMPR1B among reproduction-related candidate genes, providing additional evidence for the involvement of these genes in prolificacy (Sun et al., 2024). Moreover, SNPs in BMPR1B have previously been associated with litter size in plateau-type Tibetan sheep, indicating that reproductive performance in this population can be influenced by variation in major fecundity-related genes (Sun et al., 2023).
       
In the present study, ewes with the AB genotype at the GDF9 c.1111G>A locus had a significantly higher mean litter size than those with the AA genotype (1.85±0.18 vs. 1.52±0.15; P= 0.035). This result suggests a potential association between the c.1111G>A polymorphism and litter size in the examined plateau-type Tibetan sheep population. Similar genotype-dependent associations have recently been reported for GDF9 variants in other sheep populations. Yang et al. (2026) identified several SNPs in the GDF9 promoter region of Australian-Hu sheep that were significantly associated with lambing rate, with some heterozygous genotypes showing higher reproductive performance. The biological mechanism underlying the association observed in the present study remains unclear. Because follicular development, ovulation rate, hormone concentrations and GDF9 expression were not directly measured, the present results should not be interpreted as evidence that the c.1111G>A mutation directly alters ovulation or represents an adaptive response to the high-altitude environment. Further studies using larger populations and functional experiments are required to clarify the biological effect of this locus.
 
Significance and prospects for genetic breeding of plateau-type tibetan sheep
 
The PCR-SSCP method established in the present study provides a practical approach for preliminary screening of the GDF9 c.1111G>A polymorphism in plateau-type Tibetan sheep. The observed association between this locus and litter size suggests that it may have potential value as a candidate molecular marker for reproductive traits. However, the current evidence is not sufficient to support its direct application in marker-assisted selection because the association was identified in a relatively small sample from a single flock. Validation in larger and independent plateau-type Tibetan sheep populations is required before the locus can be considered for breeding applications.
       
Recent studies have demonstrated that reproductive performance in Tibetan sheep is regulated by multiple genes and molecular pathways. Sun et al. (2023) identified BMPR1B polymorphisms associated with litter size in plateau-type Tibetan sheep, while transcriptomic analysis of Tibetan sheep ovaries identified reproduction-related genes including GDF9, BMP15 and BMPR1B (Sun et al., 2024). These findings indicate that litter size is a complex trait that is unlikely to be adequately explained by a single candidate locus. Accordingly, future studies should integrate GDF9 with other major fecundity-related genes and evaluate their combined effects on reproductive traits. This polygenic architecture is also supported by recent sheep studies showing associations between litter size and variation in BMPR1B and other reproductive candidate genes (Zhang et al., 2022; Liu et al., 2024; Ren et al., 2024).
       
Recent research in Australian-Hu sheep has shown that multiple GDF9 promoter SNPs are significantly associated with lambing rate and that some variants can influence promoter transcriptional activity, highlighting the importance of functional validation when evaluating candidate reproductive markers (Yang et al., 2026). Future research on the c.1111G>A locus should expand the sample size, include animals from multiple independent flocks or populations and incorporate functional analyses such as gene expression, ovarian follicular development and relevant reproductive phenotypes. Such studies will help determine whether this polymorphism can serve as a reliable molecular marker for reproductive breeding in plateau-type Tibetan sheep.

This study established a PCR-SSCP-based method for detecting the GDF9 c.1111G>A polymorphism in plateau-type Tibetan sheep. Under the optimized PCR and SSCP conditions, clear and reproducible electrophoretic profiles were obtained and the identified SNP was further confirmed by sequencing. Two genotypes, AA and AB, were detected in the examined population, whereas the BB genotype was not observed. The AB genotype was associated with a significantly higher mean litter size than the AA genotype, suggesting that the c.1111G>A polymorphism may be a potential candidate marker for reproductive traits in plateau-type Tibetan sheep. However, given the relatively small sample size and the use of animals from a single flock, this association should be considered preliminary. Further validation in larger and independent populations, together with functional studies, is required before this locus can be considered for application in molecular breeding.

This study was supported by the National Modern Agricultural Industry Technology System Project (CARS-39-35) and the Applied Basic Research Project of the Department of Science and Technology of Qinghai Province (2025-ZJ-719).
 
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.

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.