Method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by PCR-SSCP and application thereof

Abstract

The application provides a method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP, taking whole genome DNA of sheep as a template, amplifying INSL3 gene fragments by PCR, then denaturing into single-stranded DNA, and then detecting and identifying the single nucleotide polymorphism of the 120th nucleotide of the sheep INSL3 gene by polyacrylamide gel electrophoresis, which can simply, rapidly, low-cost and accurately identify the single nucleotide polymorphism of the gene. The application carries out correlation analysis on the single nucleotide polymorphism of the sheep INSL3 gene and the reproductive traits, verifies that the polymorphism of the INSL3 gene can affect seasonal reproduction of different breeds of sheep, and further affect the lambing number, and shows that the single nucleotide polymorphism of the INSL3 gene can be used as a molecular genetic marker related to the traits of year-round estrus and high fertility of sheep, and is used for assisted selection breeding to improve the reproductive traits of sheep.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics technology, specifically relating to a method for rapid detection of single nucleotide polymorphisms in sheep INSL3 gene using PCR-SSCP and its application. Background Technology

[0002] Sheep are one of my country's important agricultural economic animals, and their reproductive efficiency is a key driver of profitability. Developing new strategies to improve reproductive efficiency has always been a focus of research. Xinjiang, one of my country's pastoral areas, possesses unique geographical advantages and dietary habits that provide ideal conditions for the development of sheep farming. Through long-term livestock farming practices, Xinjiang's local residents have developed a nomadic grazing tradition, preferring to raise local breeds that are highly adaptable, tolerant of roughage, and fast-growing. For example, the Kazakh sheep, a distinctive breed from northern Xinjiang, grows rapidly and is highly adaptable to different production areas; however, its seasonal estrus, ovulation, and lambing are major reasons for its low efficiency. The Dolan sheep, a distinctive breed from southern Xinjiang, is characterized by its large size, high meat yield, high reproductive rate, and genetic stability. The Cele black sheep, another distinctive breed from southern Xinjiang, is known for its year-round estrus and high reproductive rate, averaging 2-3 lambs per litter, and is one of the important breeds in Xinjiang's sheep farming industry. In recent years, the sheep industry has experienced rapid growth in both scale and efficiency. Lamb birth rate is one of the most important traits determining the profitability of sheep farming and is the foundation for high sheep productivity. Therefore, studying the relationship between high fertility genes and reproductive traits in Xinjiang local sheep breeds, and understanding the genetic mechanisms of these genes during reproduction, is crucial for breed improvement and selection.

[0003] Reproductive traits are generally considered the most important indicator among the three major economic traits in sheep, regulated by both external environment and internal genes, with low to moderate heritability. The INSL3 gene contains two exons and encodes a protein called fetal testosterone, which plays a crucial role during embryonic development, particularly in testicular development and descent. Some studies have suggested that INSL3 may be an important candidate gene influencing sheep reproductive traits. The testosterone encoded by the INSL3 gene has a significant impact on sheep reproductive traits, including testicular development, spermatogenesis, sex hormone levels, and estrus. Therefore, studying the association between the sheep INSL3 gene and reproductive traits is of great significance for further understanding sheep reproductive mechanisms and improving production performance.

[0004] Single nucleotide polymorphisms (SNPs) are important molecular genetic markers, representing DNA sequence polymorphisms caused by variations in a single nucleotide in the genome, including base transitions, insertions, and deletions. The basic principle of PCR-SSCP technology is that the DNA fragment amplified by PCR is denatured into single-stranded DNA. During electrophoresis in a neutral polyacrylamide gel, single-stranded DNA forms different three-dimensional conformations, which directly affect the migration rate. Even single-stranded DNA of the same length, differing by only a single base in their nucleotide sequence, can produce different three-dimensional conformations, resulting in different migration rates and different migration bands. Therefore, PCR-SSCP technology can be used to detect the presence of SNP sites in the genome and accurately identify the genotype of SNP sites. Single-strand conformation polymorphisms (SSCPs) are now widely recognized as genetic markers for use in breeding. This method not only possesses the accuracy of DNA sequencing but also overcomes the disadvantages of high cost, cumbersome operation, and false positives, and it has no special requirements for the detected sequence sites.

[0005] Currently, no studies have been reported on single nucleotide polymorphisms in the sheep INSL3 gene. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a method for rapid detection of single nucleotide polymorphisms (SNPs) in the INSL3 gene of sheep using PCR-SSCP and its application. This invention utilizes DNA pooling sequencing and PCR-SSCP technology to detect SNPs in the INSL3 gene. Through genetic variation studies of the INSL3 gene in three sheep populations with different reproductive traits in Xinjiang—Cele black sheep, Kazakh sheep, and Dolan sheep—genetic variation of the INSL3 gene was discovered, and the relationship between reproductive traits and these genes was analyzed, laying the foundation for finding genetic markers for reproductive traits in different sheep breeds.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for rapid detection of single nucleotide polymorphisms (SNPs) in the sheep INSL3 gene using PCR-SSCP is disclosed. The method comprises: using whole-genome DNA of the sheep as a template and primer pair P as primers, PCR amplification of the sheep INSL3 gene fragment; denaturing the PCR-amplified fragment into single-stranded DNA; and then detecting the fragment by polyacrylamide gel electrophoresis. The genotype of the SNP site on the sheep INSL3 gene is determined based on the gel electrophoresis results. The SNP in the sheep INSL3 gene is defined as the A>T mutation at position 120 of the sheep INSL3 gene.

[0009] The nucleotide sequence of primer pair P is as follows:

[0010] Forward primer F: SEQ ID NO.1,

[0011] Reverse primer R: SEQ ID NO.2.

[0012] Preferably, the PCR amplification reaction system consists of: 0.5 μL DNA, 0.5 μL each of forward and reverse primers, 7.5 μL PCR Mix and 6 μL ddH2O, for a total volume of 15 μL; the PCR amplification reaction program is: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles.

[0013] Preferably, the polyacrylamide gel electrophoresis detection conditions are: 140V for 10 min, followed by 120V for 10 h.

[0014] Preferably, the sheep INSL3 gene single nucleotide polymorphism is expressed as three genotypes: AA, AT, and TT, among which AT is the dominant genotype, which is beneficial to improving the sheep's reproductive traits.

[0015] This invention also provides a method for rapid detection of single nucleotide polymorphisms in the INSL3 gene of sheep using PCR-SSCP, which is applied to sheep reproductive trait-related assisted selection and molecular breeding. The sheep are selected from Cele black sheep, Kazakh sheep and Dolan sheep from Xinjiang.

[0016] This invention, by adopting the above technical solutions, has significant technical effects:

[0017] 1. This invention provides a method for rapid detection of single nucleotide polymorphisms (SNPs) in the sheep INSL3 gene using PCR-SSCP. Utilizing pooled DNA sequencing and PCR-SSCP technology, the SNPs of the INSL3 gene were detected in three sheep populations with different reproductive traits in Xinjiang: Cele Black Sheep, Kazakh Sheep, and Dolan Sheep. A mutation of A>T was found at position 120 of the sheep INSL3 genome. The sheep INSL3 gene SNPs exhibited three genotypes: AA, AT, and TT. This method provides a simple, rapid, low-cost, and highly accurate method for identifying gene SNPs.

[0018] 2. This invention conducts an association analysis between the single nucleotide polymorphism (SNP) of the INSL3 gene in sheep and reproductive traits. It was found that AT is the dominant genotype in three breeds: Cele Black Sheep, Kazakh Sheep, and Dolan Sheep. Furthermore, the INSL3 gene SNP of these three breeds is in Hardy-Weinberg equilibrium. The experimental results verify that the INSL3 gene SNP can affect the seasonal estrus and lambing number of different sheep breeds, demonstrating that the INSL3 gene SNP is correlated with reproductive performance. It can be used as a molecular genetic marker related to the annual estrus and lambing number traits in sheep, and can be used to assist in selective breeding to improve the reproductive traits of sheep.

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 This is the PCR amplification result of the sheep INSL3 genome at position 120 of the present invention;

[0021] Figure 2 This is a sequencing peak diagram of the PCR product of the sheep INSL3 genome at position 120 of the present invention;

[0022] Figure 3 This is the PCR-SSCP gel electrophoresis result of the sheep INSL3 genome genotype at position 120 of the present invention;

[0023] Figure 4 This invention relates to the tertiary structure of the protein after mutation of the INSL3 gene. Detailed Implementation

[0024] This invention designs primers based on the sheep INSL3 genome sequence and uses genomic DNA pools from three sheep breeds in Xinjiang—Cele Black Sheep, Kazakh Sheep, and Dolan Sheep—as templates for PCR amplification. Sequencing of the products yields a partial sequence of the sheep INSL3 gene. Comparison with the reference sequence published on NCBI reveals an A>T mutation at position 120 of the sheep INSL3 genome. This mutation site is located in the exon region of the sheep INSL3 gene, producing a synonymous mutation. Association analysis between this mutation and reproductive traits identifies it as a molecular genetic marker associated with sheep reproductive characteristics. The PCR-SSCP method is then used to detect this mutation.

[0025] 1. DNA Sample Collection

[0026] A total of 374 genomic DNA samples were obtained from venous blood collection from healthy sheep in Xinjiang. Specifically, whole blood was collected from 114 Cele Black Sheep ewes aged 4 years and weighing 47±1.36 kg at a Cele Black Sheep farm and preserved in anticoagulant tubes; whole blood was collected from 126 Kazakh Sheep ewes aged 4 years and weighing 47±1.36 kg at a Kazakh Sheep farm and preserved in anticoagulant tubes; and whole blood was collected from 134 Dolan Sheep ewes aged 4 years and weighing 47±1.36 kg at a Dolan Sheep farm and preserved in anticoagulant tubes. All sheep were housed under the same feeding conditions. Genomic DNA was extracted from the blood samples using a DNA isolation kit (DP210831, Tiangen Biotech (Beijing) Co., Ltd.).

[0027] 2. Primer design

[0028] To amplify sheep INSL3, primer pairs were designed based on reported sheep gene sequences. Primer information is shown in Table 1.

[0029] Table 1 Primer sequences

[0030]

[0031] 3. Single-strand conformation polymorphism (PCR-SSCP) analysis

[0032] Using sheep whole genome DNA as a template and primer pair P as primers, the sheep INSL3 gene fragment was amplified by PCR. The PCR amplification reaction system (15 μL) consisted of: 0.5 μL DNA, 0.5 μL each of forward and reverse primers, 7.5 μL PCR Mix and 6 μL ddH2O. The PCR amplification reaction program was: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles.

[0033] The PCR amplification products were purified and sequenced. The PCR amplification results and the sequencing peak diagrams of the PCR products are shown below. Figure 1 and Figure 2 Analysis of the sequencing peaks revealed that two different peaks at the same site indicated a single nucleotide mutation. Two detection results, A and T, appeared at position 120bp of the target fragment of the sheep INSL3 gene, indicating the SNP polymorphism of the sheep INSL3 gene. This site represents either an A or T base polymorphism.

[0034] PCR amplification was performed using PCR-SSCP technology. After verifying that the PCR product met the expected results, the PCR product was denatured to produce single-stranded DNA. Then, a suitable concentration of polyacrylamide gel was prepared according to the fragment size, and the results were detected by polyacrylamide gel electrophoresis (140V, 10min, followed by 120V, 10h). The genotype of the single nucleotide polymorphism site on the sheep INSL3 gene was determined based on the gel electrophoresis results. Figure 3 These are the results of PCR-SSCP gel electrophoresis. In the figure, 15 represents the AA genotype, 16 and 17 represent the AT genotype, and 18 represents the TT genotype.

[0035] PCR amplification and sequencing results in the DNA mixing pool showed that the size of the PCR products was consistent with the expected results, with good specificity. One single nucleotide polymorphism (SNP) was detected using the SSCP method: an A>T mutation occurred at position 120 of the INSL3 gene, with three unique genotypes: AA, AT, and TT. The reliability of the single nucleotide polymorphism (SNP) was verified by PCR-SSCP technology.

[0036] 4. Prediction of INSL3 gene SNP protein structure

[0037] DNA sequences were translated into amino acid sequences online using Novopr. The resulting amino acid sequences were then used to predict the tertiary structure of proteins after mutations in exon regions using online software (server: https: / / swissmodel.expasy.org / interactive / PU4cmH / templates / ). The results showed that the INSL3 gene sequence had an A120T synonymous mutation, but its amino acid structure remained unchanged. Figure 4 ).

[0038] 5. Differences in INSL3 genotypes among different sheep breeds

[0039] The gene frequency, genotype frequency, heterozygosity, and homozygosity of gene SNP loci were calculated using SPSS software. The chi-square test was used to check whether the genotype of each mutation locus conformed to Hardy-Weinberg equilibrium, and the differences in genotypes of candidate loci among different varieties were explored.

[0040] Genetic polymorphism analysis of INSL3 amplification sites in different sheep breeds was performed, and the results are shown in Table 2. The genotype frequency, allele frequency, heterozygosity (H), effective allele number (Ne), polymorphism information content (PIC), and Shannon information index (S) of INSL3 indicate that AA, AT, and TT genotypes exist in all three sheep breeds. The genotype frequency trends of Dolan sheep and Cele black sheep are similar, and the PIC of the two breeds is the same. AT is the dominant genotype. In the Kazakh sheep population, AA is the dominant genotype. A is the dominant allele in all three breeds, and INSL3 (A120T) is in Hardy-Weinberg equilibrium (p>0.05).

[0041] Table 2. Population genetic analysis of INSL3 gene in different sheep breeds.

[0042]

[0043] Abbreviations: MAF: minor allele frequency, H: heterozygosity, Ne: effective allele count, PIC: polymorphism information content, S: Shannon index.

[0044] 6. Relationship between INSL3 gene and litter size and seasonal breeding

[0045] Logistic regression model was used to analyze the association between different genotypes and number of lambs born: Y ijkl =μ+S i +P j +G k +F l +S i ×G k +G j ×P k +S i ×P j +e ijkl , where Y ijkl The phenotypic value of the number of lambs born; μ is the population mean; S i This is a fixed effect for the i-th lambing season (i = 1, 2, 3, 4); P j This is a fixed effect of the j-th parity (j = 1, 2, 3); G k It is the fixed effect of the k-th genotype (k = 1, 2, 3); F l This is the fixed effect of the first farm (1 = 1, 2, 3); S i ×G k It is an interaction between season and genotype; G j ×P k It is the interaction between parity and genotype; S i ×P j It is the interaction between seasons and odd / even; eijkl Random residuals were used. The results are shown in Table 3. Association analysis revealed that in Dolan sheep, individuals with the AA and AT genotypes had significantly higher litter sizes than those with the TT genotype, while in Cele black sheep, individuals with the AT genotype had significantly higher litter sizes than those with the AA and TT genotypes. Therefore, for INSL3 A120T, the AA and AT genotypes are favorable for Dolan sheep, and the AT genotype is favorable for Cele black sheep. Furthermore, in Dolan sheep, the AA and AT genotypes were associated with the highest litter size.

[0046] Furthermore, a logistic regression model was used to analyze the relationship between polymorphism and seasonal estrus: Y = μ + G + S + B + e, where Y is the phenotypic value of estrus, μ is the population mean, G is the fixed effect of genotype, S is the fixed effect of season, B is the fixed effect of breed, and e is the random residual. All data are expressed as mean ± standard error. The results are shown in Table 3. The number of lambs produced by INSL3(A120T) showed the same trend in the three breeds, with AT being the dominant genotype.

[0047] Table 3. Correlation analysis of INSL3 genotype with litter size and seasonal estrus.

[0048]

[0049] This invention discovered that AT is the dominant genotype for three breeds: Cele Black Sheep, Kazakh Sheep, and Dolan Sheep. Furthermore, the INSL3 gene single nucleotide polymorphism in these three breeds is in Hardy-Weinberg equilibrium. The experimental results verified that INSL3 gene polymorphism can affect the seasonal reproduction of different sheep breeds, thereby affecting the number of lambs born. This demonstrates that INSL3 gene single nucleotide polymorphism is correlated with reproductive performance and can serve as a molecular genetic marker associated with year-round estrus and high fertility in sheep, which can be used to assist in selective breeding to improve the reproductive traits of sheep.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. An application of a method for rapid detection of SNP sites in the sheep INSL3 gene using PCR-SSCP in marker-assisted breeding of sheep litter size, characterized in that... Using the whole genome DNA of the sheep to be tested as a template and primer pair P as primer, the sheep INSL3 gene fragment was amplified by PCR. The PCR-amplified fragment was denatured into single-stranded DNA and then detected by polyacrylamide gel electrophoresis. The genotype of the SNP site on the sheep INSL3 gene was determined based on the gel electrophoresis results. The SNP site of the sheep INSL3 gene was the 120th position of the PCR-amplified fragment, which had an A>T mutation. The sheep were either Dolan sheep or Cele black sheep. The nucleotide sequence of primer pair P is as follows: Forward primer F: SEQ ID NO.1, Reverse primer R: SEQ ID NO.2; In Dolan sheep, individuals with the AA and AT genotypes at the SNP loci had significantly higher lambing numbers than those with the TT genotype. In Cele black sheep, individuals with the AT genotype at the SNP loci had significantly higher lambing numbers than those with the AA and TT genotypes.

2. The application according to claim 1, characterized in that, The PCR amplification reaction system consisted of: 0.5 μL DNA, 0.5 μL each of forward and reverse primers, 7.5 μL PCR Mix and 6 μL ddH2O, for a total volume of 15 μL. The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles.

3. The application according to claim 1, characterized in that, The polyacrylamide gel electrophoresis detection conditions were: 140V for 10 min, followed by 120V for 10 h.

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