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Article

Investigation of a Rare CSN1S101 Allele in Goats Using Allele-Specific PCR and Mathematical Expectation Analysis

by
Jingxuan Wang
1,†,
Yuta Yang
1,†,
Yuhuan Gao
1,
Ebadu Areb
1,
Xianyong Lan
1,
Haijing Zhu
2 and
Chuanying Pan
1,*
1
College of Animal Science and Technology, Northwest A&F University, Yangling, Xianyang 712100, China
2
College of Life Sciences, Yulin University, Yulin 719000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Animals 2026, 16(5), 730; https://doi.org/10.3390/ani16050730
Submission received: 14 January 2026 / Revised: 20 February 2026 / Accepted: 24 February 2026 / Published: 26 February 2026
(This article belongs to the Special Issue Genetics and Breeding for Enhancing Production Traits in Ruminants)
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Simple Summary

The casein alpha s1 (CSN1S1) gene is a critical milk protein locus in goats, characterized by extensive genetic polymorphism. The CSN1S101 allele is a key ‘null’ variant. However, its distribution across diverse breeds and large populations remains unclear. Furthermore, its associations with economic traits have not been fully elucidated. In this study, a cost-effective genotyping approach was employed to screen 2319 goats from 11 Chinese breeds for the CSN1S101 allele. Our results show that the “1” allele predominates across all populations, whereas the “0” allele occurs at a low frequency, indicating limited genetic diversity at this locus. Association analysis revealed that CSN1S101 genotypes are significantly correlated with body length, third-parity litter size, and average litter size, specifically in Laoshan dairy goats. This study provides important evidence for the association of CSN1S101 genetic variation within goat populations.

Abstract

The casein alpha s1 (CSN1S1) gene plays an important role in milk protein synthesis and has been associated with milk quality and calcium-related physiological processes in mammals. To date, 19 CSN1S1 variants have been reported. Among them, the CSN1S101 allele has been described as a key “null” variant that may influence goat milk production traits through its effect on CSN1S1 gene expression. However, the association between this allele and economically important traits in goats remains poorly understood. Therefore, the present study aimed to investigate the CSN1S101 allele in goats using economical and rapid approaches, including mathematical expectation (ME) analysis and allele-specific PCR (AS-PCR), and to evaluate its association with economic traits in Chinese goat breeds. A total of 2319 goats from 11 breeds were analyzed. Compared with the “0” allele, the “1” allele showed a higher frequency across all populations and exhibited low genetic diversity. Significant associations were detected between CSN1S101 genotypes and body length, third parity, and average kidding number in Laoshan dairy goats (p < 0.05). In addition, the low-frequency distribution of the CSN1S1 “0” allele was further confirmed. Overall, this study provides population-based association evidence supporting the consideration of CSN1S101 genetic variation in goat breeding programs.

1. Introduction

Casein is a unique phosphoprotein in milk that contains high levels of calcium and phosphorus. In goats (Capra hircus), caseins consist of four polypeptide chains: αs1, αs2, β, and k caseins [1]. The CSN genes encoding casein are linked as CSN1S1-CSN1S2-CSN2-CSN3, and the gene cluster spans about a 250 kb region on goat chromosome 6 [2,3]. Polymorphisms in these four genes are associated with milk yield and quality traits (fat, protein, and total solids), regulation of milk enzyme activity and edible suitability, body measurement traits, and reproductive traits [4,5,6].
As we know, the αs1 -casein (CSN1S1) is one of four caseins synthesized by the lactating mammary gland. The goat CSN1S1 gene is generally polymorphic; at least 19 CSN1S1 protein variants have been identified in goats and used for breed characterization, biodiversity, and phylogenetic research. Detailed information on the identified alleles is summarized in Table 1. Most of the mutations contributing to CSN1S1 allele differentiation have been characterized. The B1 allele is considered the ancestral form and has given rise to two major lineages: type A (A, A2, A3, 01, 02, I, G, and H) and type B (B2, B3, B4, C, E, F, and L) [7]. Previous studies have suggested that interallelic recombination events between type-A and type-B alleles may have contributed to the formation of the CSN1S1 N and M alleles [8]. Certain alleles, such as the F allele, are distinguished by specific intronic insertions or deletions [9,10]. Molecular approaches including PCR-SSCP have been widely used to identify alleles such as N and F, and previous studies reported associations with milk yield and composition in Chinese dairy goats [11]. The 02 allele has been described as a null allele containing a large insertion that has not yet been fully characterized. In addition, alleles 01 and 02 are called ‘null’ alleles and cause the deficiency of αs1-casein [12]. An 11 bp indel of this gene was associated with litter size and body measurement traits in goats, suggesting a potential link between CSN1S1 polymorphisms and multiple traits [6,13]. However, the association between different CSN1S1 alleles and body measurements and reproductive traits of goats has not been reported. The low-frequency CSN1S101 allele of the goat CSN1S1 gene was mentioned in previous studies, and the mutational origin of this allele is the large deletion of a DNA segment of approximately 8.5 kb, starting from the 181st nucleotide of intron 12 [14]. Although the CSN1S101 allele is considered a null allele and has multiple functions such as goat milk allergenicity [15]. Milk obtained from goats carrying the “null” allele may be more suitable for specific nutritional or dietary purposes, specific transformation technology processes, or for “humanized” milk production [16]. Given the diversity of CSN1S1 gene functions, its influence on growth and reproductive traits requires further exploration. Previous studies have shown that the polymorphism of the CSN1S101 allele in goat breeds [17]. Thus, we detected this rare mutation in 11 goat breeds by combining the allele-specific polymerase chain reaction method (AS-PCR) and the mathematical expectation (ME) method [18] and explored the relationship between mutation and its association with growth and reproductive traits.
In this study, we described an economical and rapid method for analyzing the goat CSN1S101 allele and found a significant association between different genotypes of the CSN1S101 allele and goat growth and reproductive traits. Therefore, this study provides population-based association evidence that may inform future investigations into the role of CSN1S101 genetic variation in goat breeding programs.

2. Materials and Methods

2.1. Samples and Data Collection

In this study, 2319 goats representing 11 breeds were used. Samples of Shaanbei white cashmere goats (SBWC) were collected from Yulin, Shaanxi Province. Guizhou Heima goat (GZHM) and Nubia goat (NG) samples were collected from Guizhou province. Guanzhong (GZ) and Xinong Saanen (SA) dairy goats were collected from Qianyang County in Shaanxi Province. Inner Mongolia white cashmere goats (IMWC) were collected from Ordos City in the Inner Mongolia Autonomous Region. Goats of the same breed were raised and managed under identical conditions, and individuals included in this study were randomly selected. Body measurement traits were recorded following the protocol described by Zhang et al. [24]. Sample collection and storage procedures were performed according to the previously reported method [6].

2.2. DNA Isolation and Primer Design

Genomic DNA was extracted from the collected ear tissue samples using the high-salt extraction method [25]. A Nanodrop 2000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the DNA purity and quality. Subsequently double-distilled water was added to dilute the extracted DNA to 20 ng/μL and stored at −20 °C. A set of allele-specific primers was designed based on previously published methods to amplify a region spanning part of exon 12 and the adjacent intron 12 of the goat CSN1S1 gene, which contains the deletion characteristic of the CSN1S101 null allele [17]. The primers used were: goat-CSN1S1-AS-a (5′-CCCCAGCTGGTAATGTTTTA-3′), goat-CSN1S1-AS-b (5′-GGTCCATCAATTCCCTGTGT-3′), and goat-CSN1S1-AS-c (5′-TGTATGGATCCCTGATTCCTTC-3′). The amplified fragment is approximately 249 bp for the CSN1S101 allele and 281 bp for the reference allele, allowing clear discrimination of homozygous, heterozygous, and reference genotypes. Primer positions relative to the gene structure are illustrated in a schematic diagram. The PCR was performed in a 20 μL reaction mixture containing 2 μL of DNA solution (20 ng) goat genomic DNA (constructed from four different individuals), 10 μL of 2× Eco Taq PCR Super mix (Vazyme Biotech Co., Ltd., Nanjing, China; Cat# No. P222-01), 0.6 μL of forward primer, 0.4 μL of reverse primer, and 6.6 μL of double distilled. The protocols for the PCR reaction mixture and amplification conditions used in the present study were based on Yang et al. [26]. A 3.5% agarose gel was used for electrophoresis of the PCR products to identify the indel locus [27].

2.3. Statistical Analysis

Previously reported methods were used to examine the Hardy–Weinberg equilibrium (HWE) and population-genetic parameters, such as homozygosity (Ho), effective allele numbers (Ne), and polymorphism information content (PIC) [28]. The association between the goat CSN1S101 allele and economic traits was analyzed using t-test on SPSS software (version 24.0). A linear model of the relationship between goat genotypes and each growth trait was constructed using Hui’s method [29].

3. Results

3.1. Identification of Indel Variations and Genotyping

The AS-PCR amplification results showed that there were two genotypes in the goat population: the A1A1 genotype showed one band (281 bp), and the A1A0 genotypes showed two bands (281 and 249 bp) (Figure 1). However, only the A1A1 genotype was detected in IMWC, GB, BJ, MT, GW, SBWC, and NG goats (Table 2).

3.2. Genetic Parameter Analysis

The frequency of the A1 allele ranged between 0.946 and 1.000, and this locus was in Hardy–Weinberg equilibrium in all populations (p > 0.05). The genotypic distribution among the 11 goat breeds was determined by calculating the genetic parameters associated with the CSN1S101 allele, such as genotype and allele frequencies (Table 2). Compared with the “0” allele, the “1” allele had a higher frequency in the 11 goat breeds. Based on the PIC values, this allele showed low genetic diversity in all breeds.

3.3. Association Analysis of Genotypes and Body Measurement Traits

The association between the CSN1S101 allele and economic traits in Laoshan dairy goats is shown in Table 3 and Table 4. The CSN1S101 allele was highly associated with third parity and average kidding number (p < 0.05, Table 3). A doe with A1A0 genotype produces approximately 0.6 more kids per parity (2.25 vs. 1.61) represent a major economic benefit through more replacement stock. Similarly, the CSN1S101 allele was also associated with body length (p < 0.05, Table 4). For all other traits (weight, height, chest, cannon), the A1A0 genotype had numerically higher averages than A1A1 but statistically non-significant.

4. Discussion

In this study, the CSN1S101 polymorphism was systematically characterized across multiple goat populations using AS-PCR and ME methods. Only two genotypes (A1A1 and A1A0) were detected, and the A0 allele was observed at a very low frequency and only in a limited number of breeds. This distribution pattern indicates that CSN1S101 is a low-polymorphism locus in goats, which is consistent with previous reports describing the rarity of the A0 allele in different geographic and production populations [13,17,30,31].
The investigation of genetic variation in the CSN1S1 gene and its association with production traits is of considerable interest. Previous studies have demonstrated that polymorphisms in CSN1S1 are associated with variation in milk casein content as well as milk structure and nutritional properties. To date, ruminants—including cattle, sheep, and goats—remain the primary source of milk for human consumption worldwide, and most studies on CSN1S1 have therefore focused on these species. In cattle, ten CSN1S1 protein variants (A, B, C, D, E, F, G, H, I, and J) have been reported, and several alleles have been associated with differences in milk composition traits, such as milk fat percentage and CSN1S1 content [32,33]. In sheep, seven CSN1S1 phenotypes (A, B, C, D, E, F, and X) have been identified [34]. Beyond milk composition, CSN1S1 polymorphisms have also been examined in association studies involving other economically relevant traits in livestock. For instance, CSN1S1 polymorphisms have been associated with lower yearling weights in cattle [35]. In goats, CSN1S1 polymorphisms have also been linked to reproductive and growth traits [14,24]. However, the biological basis of these associations remains unclear. Overall, existing evidence indicates that genetic variation at the CSN1S1 locus is primarily relevant to milk-related traits, while reported associations with non-milk traits should be considered descriptive and population-specific rather than indicative of direct functional effects.
In the present study, we focused specifically on the CSN1S101 allele across different goat populations. Methods including ME and AS-PCR were established for reliable identification of the 01 allele, and the reported mutation frequency of this allele in other goat populations is summarized in Table 5. Notably, while the frequency of the CSN1S101 allele varies across breeds, it reaches an exceptionally high level (approximately 70–73%) in Norwegian Dairy goats, significantly impacting milk composition and technological properties [36,37]. Genetic parameter analysis revealed that the CSN1S1 locus adheres to the Hardy–Weinberg equilibrium (HWE) across all studied populations, with PIC values indicating a low genetic diversity. These results suggest a relatively stable allelic distribution and imply that strong directional selection is unlikely to be acting on this locus. However, the limited polymorphism observed necessitates caution when interpreting downstream association analyses, particularly concerning rare genotypes. Association analysis demonstrated that the CSN1S101 allele is significantly correlated with the third-parity litter size and body length in Laoshan dairy goats, whereas no significant associations were detected for other morphological traits. Notably, these associations were inconsistent across breeds and traits, highlighting a clear population- and trait-specific pattern. The absence of significant effects on most growth-related parameters suggests that CSN1S1 polymorphisms do not exert a broad influence on overall body conformation. Reproductive performance and body measurements are complex quantitative traits governed by polygenic inheritance and environmental-management interactions. The parity-specific associations observed here further reflect the complexity of reproductive traits, potentially influenced by sample structure, breeding history, or management practices. Consequently, the identified associations should be interpreted as localized statistical correlations rather than evidence of a universal or direct genetic effect.
From a methodological perspective, this study establishes a practical framework for the detection and population-level screening of the CSN1S1 allele in goats. The integrated application of AS-PCR and ME methods ensures reliable genotype differentiation across diverse breeds, facilitating future investigations into CSN1S1 polymorphisms within various genetic backgrounds. However, several limitations must be acknowledged. First, the low frequency of the A0 allele constrained the statistical power of our association analyses, particularly in breeds where only the A1A1 genotype was present. Second, this study focused on a single locus without integrating functional or expression data. Finally, environmental and management factors, which can influence reproductive performance and growth traits [40], should be considered in future investigations as their potential effects were not explicitly modeled here. These constraints underscore the necessity for further validation in independent cohorts and integrated omics research to fully elucidate the biological significance of CSN1S1 variations.
In summary, this study systematically characterized the genetic variations in CSN1S1 across Chinese goat populations and elucidated population-specific associations with key growth and reproductive traits. While these findings are preliminary and descriptive in nature, they expand the current understanding of CSN1S1 polymorphisms and provide a valuable foundation for subsequent functional validation.

5. Conclusions

AS-PCR and mathematical expectation (ME) methods can be used to rapidly and efficiently detect goat CSN1S101 allelic mutations. The low-frequency characteristics of the CSN1S101 allele are universal in the goat breeds. The CSN1S101 allele shows population- and breed-specific associations with selected growth and reproductive traits in Laoshan dairy goats.

Author Contributions

Conceptualization, C.P. and X.L.; methodology, J.W. and Y.Y.; formal analysis, J.W. and Y.Y.; investigation, J.W., Y.G. and E.A.; data collection, J.W., Y.Y., Y.G. and X.L.; writing—original draft preparation, J.W. and Y.Y.; writing—review and editing, J.W., Y.Y., E.A. and X.L.; supervision, C.P.; project administration, C.P. and H.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2022YFD1300200) and National Natural Science Foundation (No. 32272850); China Agriculture Research System of MOF and MARA (CARS-34), the National Innovation Project for Undergraduate of Northwest A&F University (No. XN2025004117), and the University Innovation Project for Undergraduate of Northwest A&F University (XN2025001068; XN2026004160).

Institutional Review Board Statement

All animal procedures were conducted in accordance with the national guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Northwest A&F University (IACUC-NWAFU).

Informed Consent Statement

Written informed consent was obtained from the owner of the animals involved in this study.

Data Availability Statement

All data analyzed during this study are available from the corresponding author upon reasonable request.

Acknowledgments

We are grateful for the support from the Shaanbei White Cashmere goat breeding farm, Yulin City, Shaanxi Province, China. We would also like to thank Lei Qu, Hailong Yan, and Jinwang Liu from Yulin University for their help in sample collection. Thanks to Chuchao Lei of the Academy of Animal Sciences for providing the goat samples; thanks to the Guizhou Institute of Animal Husbandry and Veterinary Medicine; thanks to the Fujian Academy of Agricultural Sciences for the samples; Ruoyu Liu of Guizhou University for the samples; and Lingjiang Min of Qingdao Agricultural University.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

Sa: Saanen dairy goat; LS: Laoshan dairy goat; GZ: Guanzhong dairy goat; IMWC: Inner Mongolia white cashmere goat; GZBG: Guanzhong black goat; BJ: Banjiao goat; MT: matou goat; GW: Guizhou white goat; SBWC: Shaanbei White Cashmere Goat; GZHM: Guizhou Heima goat; AS-PCR: allele-specific polymerase chain reaction; ME: mathematical expectation.

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Animals 16 00730 g001
Figure 1. Electrophoresis of AS-PCR products of goat CSN1S1 gene. Note: Red arrows indicate the transcription orientation of genes, colored boxes represent exonic regions, the blue arrow denotes the deletion locus analyzed in this study, and the dashed line indicates the genomic distance between CSN1S1 and CSN2 (~8.5 kb).
Figure 1. Electrophoresis of AS-PCR products of goat CSN1S1 gene. Note: Red arrows indicate the transcription orientation of genes, colored boxes represent exonic regions, the blue arrow denotes the deletion locus analyzed in this study, and the dashed line indicates the genomic distance between CSN1S1 and CSN2 (~8.5 kb).
Animals 16 00730 g001
Table 1. Details on the goat CSN1S1 alleles.
Table 1. Details on the goat CSN1S1 alleles.
AllelesVariationsReferences
AGlu (77) → Gln[19]
A2uncharacterizedNA
A3uncharacterizedNA
B1Reference 199 residues[20]
B2Leu (16) → pro[21]
B3Leu (16) → pro, Arg (100) → Lys[21]
B4Leu (16) → pro, Arg (100) → Lys, Thr (195) → Ala[21]
CLeu (16) → Pro; Arg (100) → Lys; Thr (195) → Ala; His (8) → Ile[19]
DLeu (16) → Pro, deletion 59 to 69[19]
Ea 457 bp insertion within exon 19 (last untranslated exon)[22]
Fpresence of short insertions of 11 bp (CGTAATGTTTC) (nucleotides 9972–9982) and 3 bp (AAT) (nucleotides 10,639–1042) inside the 9th intron, and by the deletion of 7 bp (TTATCTA) at the 14th intron between the 14,647 and 14,648 nucleotides[9]
GGlu (77) → Gln, deletion 14 to 26[21]
HGlu (77) → Gln; Arg (1) → Lys[9]
Iunknown[23]
LLeu (16) → Pro; Arg (90) → His[23]
MSer (66) → Leu
a single nucleotide transition C → T (nucleotide 23/exon 9)		[23]
Nthe deletion of cytosine at the 23rd nucleotide of the 9th exon, without the insertion of 11 and 3 bp in the subsequent intron[8]
01the deletion of a DNA segment of about 8.5kb starting from the 181st nucleotide of the intron 12, and including the last 7 exons of the gene[9]
02A large insertion, so far uncharacterizedNA
“NA” indicates: Not Available.
Table 2. The population genetic metrics of the CSN1S1 locus for different populations.
Table 2. The population genetic metrics of the CSN1S1 locus for different populations.
TypesBreedsA1A1
Genotype		A1A0
Genotype		TotalA1/A0 AllelesHoHeNePICX2
Value
Cashmere typeIMWC689147030.985/0.0150.9690.0311.0320.030.11
SBWC1031010311.000/010100
Meat typeGZHM20012010.995/0.0050.9950.0051.0050.0050.001
NG690691.000/010100
GZBG210211.000/010100
BJ250251.000/010100
MT220221.000/010100
GW310311.000/010100
Dairy typeSa668740.946/0.0540.8980.1021.1140.0970.24
LS764800.975/0.0250.9510.0491.0510.0480.05
GZ602620.984/0.0160.9680.0321.0330.0310.02
Note: Sa = Saanen dairy goat, LS = Laoshan dairy goat, GZ = Guanzhong dairy goat, IMWC = Inner Mongolia white cashmere goat, GZBG = Guanzhong black goat, BJ = Banjiao goat, MT = matou goat; GW = Guizhou white goat; SBWC = Shaanbei White Cashmere Goat; GZHM = Guizhou heima goat; NG = Nubia goat.
Table 3. Association between different genotypes of the CSN1S1 gene with litter size (means ± SE) in Laoshan dairy goats.
Table 3. Association between different genotypes of the CSN1S1 gene with litter size (means ± SE) in Laoshan dairy goats.
Fixed EffectsA1A1 Genotype (n = 76)A1A0 Genotype (n = 4)p-Value
The third parity1.67 ± 0.10 a2.50 ± 0.29 b0.02
Average1.61 ± 0.08 a2.25 ± 0.43 b0.05
Note: Different lowercase letters (a, b) indicate significant differences at p < 0.05. “The third parity” refers to the number of kids born at the third kidding. “Average” refers to the mean number of kids per kidding.
Table 4. Association between different genotypes of CSN1S1 gene and goat economic traits (Mean ± SE) in Laoshan dairy goat.
Table 4. Association between different genotypes of CSN1S1 gene and goat economic traits (Mean ± SE) in Laoshan dairy goat.
TraitsA1A1 Genotype
(n = 76)		A1A0 Genotype
(n = 4)		p-Values
body weight (kg)47.30 ± 1.0951.00 ± 5.000.490
body height (cm)76.05 ± 0.5280.50 ± 0.500.119
body length (cm)78.29 ± 0.52 a85.50 ± 1.50 b0.014
chest circumference(cm)79.34 ± 0.5283.00 ± 0.530.215
cannon circumference (cm)9.63 ± 0.1310.00 ± 0.120.601
Note: Different lowercase letters (a, b) indicate significant differences at p < 0.05.
Table 5. The allele frequency of the CSN1S1 locus for the reported populations.
Table 5. The allele frequency of the CSN1S1 locus for the reported populations.
BreedsTotalA1/A0 AllelesReferences
GSB (White shorthaired)123
333		0.976/0.024
0.981/0.019		[12]
[12]
BSH (Brown shorthaired)45
165		0.989/0.011
0.985/0.015		[12]
[12]
Sarda goats9350.999/0.001[31]
Frisa700.893/0.107[30]
Orobica660.977/0.023[30]
Verzasca670.993/0.007[30]
Camosciata880.9890/0.011[30]
Neapolitan goats2851.000/0[7]
Alpine breeds830.990/0.010[38]
Kilis601.000/0[39]
Sanliurfa661.000/0[39]
Siirt551.000/0[39]
Norwegian dairy goat5750.263/0.737[36,37]
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Wang, J.; Yang, Y.; Gao, Y.; Areb, E.; Lan, X.; Zhu, H.; Pan, C. Investigation of a Rare CSN1S101 Allele in Goats Using Allele-Specific PCR and Mathematical Expectation Analysis. Animals 2026, 16, 730. https://doi.org/10.3390/ani16050730

AMA Style

Wang J, Yang Y, Gao Y, Areb E, Lan X, Zhu H, Pan C. Investigation of a Rare CSN1S101 Allele in Goats Using Allele-Specific PCR and Mathematical Expectation Analysis. Animals. 2026; 16(5):730. https://doi.org/10.3390/ani16050730

Chicago/Turabian Style

Wang, Jingxuan, Yuta Yang, Yuhuan Gao, Ebadu Areb, Xianyong Lan, Haijing Zhu, and Chuanying Pan. 2026. "Investigation of a Rare CSN1S101 Allele in Goats Using Allele-Specific PCR and Mathematical Expectation Analysis" Animals 16, no. 5: 730. https://doi.org/10.3390/ani16050730

APA Style

Wang, J., Yang, Y., Gao, Y., Areb, E., Lan, X., Zhu, H., & Pan, C. (2026). Investigation of a Rare CSN1S101 Allele in Goats Using Allele-Specific PCR and Mathematical Expectation Analysis. Animals, 16(5), 730. https://doi.org/10.3390/ani16050730

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