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

Abstract

The invention provides a method for rapidly detecting the single nucleotide polymorphism of a sheep INSL3 gene by using PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism), which comprises the following steps of: performing PCR amplification on an INSL3 gene segment by taking sheep whole genome DNA (deoxyribonucleic acid) as a template, then denaturating into single-stranded DNA, and identifying the 120th single nucleotide polymorphism of the sheep INSL3 gene according to a polyacrylamide gel electrophoresis detection result. The method can be used for simply and quickly identifying the single nucleotide polymorphism of the gene with low cost and high accuracy. According to the invention, association analysis is carried out on the single nucleotide polymorphism of the INSL3 gene of the sheep and reproduction traits, and the INSL3 gene polymorphism is verified to influence seasonal reproduction of different varieties of sheep and further influence the lambing number, so that the INSL3 gene single nucleotide polymorphism can be used as a molecular genetic marker related to perennial oestrus and high-fertility traits of the sheep and is used for assisted selective breeding, and the breeding efficiency of the sheep is improved. The breeding traits of the sheep are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics, and in particular relates to a method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP and application thereof. Background Art

[0002] Sheep is one of the important agricultural economic animals in my country. The reproductive efficiency of sheep is the key driving factor for profitability. Developing new strategies to improve reproductive efficiency has always been the focus of many researchers. Xinjiang is one of the pastoral areas in my country. Its unique geographical advantages and eating habits provide unique conditions for the development of sheep farming. After a long period of animal husbandry, local residents in Xinjiang have the custom of nomadic grazing and prefer to raise these local breeds with strong adaptability, tolerance to roughage, and fast growth and development. For example, Kazakh sheep is a characteristic local breed in northern Xinjiang. It grows and develops fast and has strong adaptability to the production area. However, its seasonal estrus, ovulation, and lambing are the main reasons for the low efficiency of the breeding industry; Duolang sheep is a characteristic local breed in southern Xinjiang. It has the characteristics of large size, high meat production, high reproduction rate, and genetic stability; Cele black sheep is also a characteristic local breed in southern Xinjiang. It is famous for its year-round estrus and high reproduction rate. On average, it can produce 2 to 3 lambs per litter and is in estrus all year round. It is one of the important breeds of sheep farming in Xinjiang. In recent years, the scale and benefits of the sheep industry have achieved rapid growth. The number of lambs born is one of the most important traits that determine the benefits of the sheep industry, and it is also the basis for high production and high yield of sheep. Therefore, studying the relationship between high fertility genes and reproductive traits of Xinjiang local sheep breeds and understanding the genetic mechanism of these genes in the reproduction process are important for improving and breeding varieties.

[0003] Reproductive traits are generally considered to be the most important indicators among the three major economic traits of sheep. They are regulated by the external environment and internal genes, and have low to medium heritability. The INSL3 gene contains 2 exons. INSL3 encodes a protein called fetal testosterone, which plays an important role during embryonic development, especially in the development and descent of the testicles. Some studies have suggested that INSL3 may be an important candidate gene affecting the reproductive traits of sheep. The testosterone encoded by the INSL3 gene has an important effect on the reproductive traits of sheep, including testicular development, sperm production, sex hormone levels and estrus. Therefore, studying the association between the sheep INSL3 gene and reproductive traits is of great significance for further understanding the reproductive mechanism of sheep and improving production performance.

[0004] Single nucleotide polymorphism (SNP) is an important molecular genetic marker, which is a DNA sequence polymorphism caused by a single nucleotide variation in the genome, including base conversion, insertion and deletion. The basic principle of PCR-SSCP technology is that the DNA fragments after PCR amplification are denatured into single-stranded DNA. The single-stranded DNA forms different three-dimensional conformations when electrophoresed in a neutral polyacrylamide gel, and its conformation directly affects the swimming rate. The same length of DNA single strands can produce different three-dimensional conformations with only a single base difference in the nucleotide sequence, resulting in different swimming rates and different swimming bands. Therefore, the PCR-SSCP technology can be used to detect whether there are SNP sites in the genome, and accurately identify the genotype of the SNP site. It is now recognized that single-stranded conformation polymorphism (SSCP) can be used as a genetic marker in breeding. This method not only has the accuracy of DNA sequencing, but also overcomes the disadvantages of high cost, cumbersome operation, and false positives, and there are no special requirements for detecting sequence sites.

[0005] At present, there are no reports on the single nucleotide polymorphism of sheep INSL3 gene. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for quickly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP and its application in view of the above-mentioned deficiencies in the prior art. The present invention uses DNA mixed pool sequencing and PCR-SSCP technology to detect the single nucleotide polymorphism of INSL3 gene, and studies the genetic variation of INSL3 gene of three sheep groups with different reproductive traits, namely Cele black sheep, Kazakh sheep and Duolang sheep in Xinjiang, to find the genetic variation of INSL3 gene, analyze the relationship between reproductive traits and these genes, and lay a foundation for finding genetic markers of reproductive traits of different breeds of sheep.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP, the method comprising: using the whole genome DNA of the sheep to be tested as a template and a primer pair P as a primer, PCR amplifying the sheep INSL3 gene fragment, denaturing the PCR amplified fragment into single-stranded DNA, and then detecting it by polyacrylamide gel electrophoresis, and determining the genotype of the single nucleotide polymorphism site on the sheep INSL3 gene according to the gel electrophoresis result; the single nucleotide polymorphism of the sheep INSL3 gene is the single nucleotide polymorphism of the 120th position A>T mutation of the sheep INSL3 gene;

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

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

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

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

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

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

[0015] The present invention also provides an application of a method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP in sheep reproductive trait-related assisted selection and molecular breeding, wherein the sheep are selected from Xinjiang local Cele black sheep, Kazakh sheep and Duolang sheep.

[0016] The present invention has significant technical effects due to the adoption of the above technical solution:

[0017] 1. The present invention provides a method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene by using PCR-SSCP. By using DNA mixed pool sequencing and PCR-SSCP technology, single nucleotide polymorphism (SNP) of INSL3 gene of three sheep populations with different reproductive traits, namely Cele Black Sheep, Kazakh Sheep and Duolang Sheep in Xinjiang, is detected. It is found that there is a mutation of A>T at position 120 of sheep INSL3 genome, and the single nucleotide polymorphism of sheep INSL3 gene is expressed as three genotypes, namely AA, AT and TT. The method can identify single nucleotide polymorphism of gene simply, rapidly, at low cost and with high accuracy.

[0018] 2. The present invention conducted an association analysis between the single nucleotide polymorphism of the INSL3 gene of sheep and reproductive traits, and found that AT is the dominant genotype of the three breeds of Cele Black Sheep, Kazakh Sheep and Duolang Sheep, and the single nucleotide polymorphism of the INSL3 gene of these three breeds is in Hardy-Weinberg equilibrium. The experimental results verified that the INSL3 gene polymorphism can affect the seasonal estrus and lambing number of sheep of different breeds, and proved that the single nucleotide polymorphism of the INSL3 gene has a certain correlation with reproductive performance, and can be used as a molecular genetic marker related to the perennial estrus and lambing number traits of sheep, and used to assist in selective breeding to improve the reproductive traits of sheep.

[0019] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the result of PCR amplification of the 120th genotype of the sheep INSL3 genome of the present invention;

[0021] Figure 2 It is the sequencing peak diagram of the 120th genotype PCR product of the sheep INSL3 genome of the present invention;

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

[0023] Figure 4 It is the protein tertiary structure after the INSL3 gene mutation of the present invention. DETAILED DESCRIPTION

[0024] The present invention designs primers based on the sheep INSL3 genome sequence, uses the genome DNA pools of three sheep breeds, namely, Cele Black Sheep, Kazakh Sheep and Duolang Sheep, in Xinjiang as templates, performs PCR amplification, and obtains a partial sequence of the sheep INSL3 gene by sequencing the products. Comparison with the reference sequence published on NCBI shows that there is an A>T mutation at position 120 of the sheep INSL3 genome, and the mutation site is located in the exon region of the sheep INSL3 gene, generating a synonymous mutation, and performing association analysis with the reproductive traits, determining it as a molecular genetic marker associated with the reproductive traits of sheep, and detecting the mutation using the PCR-SSCP method.

[0025] 1. DNA sample collection

[0026] 374 genomic DNA samples were obtained from venous blood samples collected from healthy sheep in Xinjiang. Among them, whole blood was collected from 114 Cele black sheep ewes aged 4 years and weighing 47±1.36 kg at the Cele black sheep breeding farm and stored in anticoagulant tubes; whole blood was collected from 126 Kazakh sheep ewes aged 4 years and weighing 47±1.36 kg at the Kazakh sheep breeding farm and stored in anticoagulant tubes; whole blood was collected from 134 Duolang sheep ewes aged 4 years and weighing 47±1.36 kg at the Duolang sheep breeding farm and stored in anticoagulant tubes. They were placed under the same breeding conditions. Genomic DNA was extracted from blood samples using a DNA isolation kit (DP210831, Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0027] 2. Primer design

[0028] To amplify sheep INSL3, a primer pair was designed based on the reported sheep gene sequence. The primer information is shown in Table 1.

[0029] Table 1 Primer sequences

[0030]

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

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

[0033] The PCR amplification products were purified and sequenced. The PCR amplification results and PCR product sequencing peaks are shown in Figure 1 and Figure 2 The sequencing peak graph was analyzed, and two different peaks at the same site indicated a single nucleotide mutation. The 120bp position of the target fragment of the sheep INSL3 gene showed two detection results, A and T, which was the SNP polymorphism of the sheep INSL3 gene screened, and the site was a base polymorphism of A or T.

[0034] The PCR amplification was performed using the PCR-SSCP technique. After the PCR product was verified to be consistent with the expected result, the PCR product was denatured to produce single-stranded DNA. Then, a polyacrylamide gel of appropriate concentration was configured according to the fragment size. The polyacrylamide gel electrophoresis was used for detection (140V, 10min, then 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 This is the result of PCR-SSCP gel electrophoresis. In the figure, 15 is the AA genotype, 16 and 17 are the AT genotypes, and 18 is the TT genotype.

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

[0036] 4. INSL3 gene SNP protein structure prediction

[0037] The DNA sequence was translated into an amino acid sequence using novopr online, and the obtained amino acid sequence was used to predict the tertiary structure of the protein after the exon region mutation through 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 did not change ( Figure 4 ).

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

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

[0040] The genetic polymorphism of the INSL3 amplification site in different sheep breeds was analyzed. 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 showed that the three sheep breeds all had AA, AT and TT genotypes. The genotype frequency trends of Duolang sheep and Cele black sheep were similar. The PIC of the two breeds was the same, and AT was the dominant genotype. In the Kazakh sheep population, AA was the dominant genotype. A was the dominant allele in the three breeds, and INSL3 (A120T) was 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 number, PIC: polymorphism information content, S: Shannon information index.

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

[0045] Logistic regression model was used to analyze the association between different genotypes and litter size: 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 is the phenotypic value of lambing size; μ is the overall mean; S i is the fixed effect of the i-th lambing season (i=1,2,3,4); P j is the fixed effect of the jth parity (j = 1, 2, 3); G k is the fixed effect of the kth genotype (k = 1, 2, 3); F l is the fixed effect of the first farm (1 = 1, 2, 3); S i ×G k is the interaction between season and genotype; G j ×P k is the interaction between parity and genotype; S i ×P j is the interaction between season and odd-even; eijkl Random residual. The results are shown in Table 3. The association analysis showed that the number of lambs born in Duolang sheep with AA and AT genotypes INSL3 A120T was significantly higher than that of TT genotype individuals, and the number of lambs born in Cele black sheep with AT genotype INSL3 A120T was significantly higher than that of AA and TT genotype individuals. Therefore, for INSL3 A120T, AA and AT genotypes are favorable genotypes for Duolang sheep, and AT genotype is a favorable genotype for Cele black sheep. In addition, in Duolang sheep, AA and AT genotypes are associated with the largest number of lambs.

[0046] In addition, the relationship between polymorphism and seasonal estrus was analyzed using a logistic regression model: Y = μ + G + S + B + e, where Y is the phenotypic value of estrus, μ is the overall 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 litter size trait of INSL3 (A120T) in the three breeds showed the same trend of change, and AT was the dominant genotype.

[0047] Table 3 Correlation analysis between INSL3 genotype, litter size and seasonal estrus

[0048]

[0049] The present invention finds that AT is the dominant genotype of three breeds, namely, Cele Black Sheep, Kazakh Sheep and Duolang Sheep, and the single nucleotide polymorphisms of INSL3 genes of these three breeds are in Hardy-Weinberg equilibrium. The experimental results verify that the INSL3 gene polymorphism can affect the seasonal reproduction of different breeds of sheep, thereby affecting the number of lambs born, and prove that the INSL3 gene single nucleotide polymorphism has a certain correlation with reproductive performance, and can be used as a molecular genetic marker related to the perennial estrus and high fertility traits of sheep, and used to assist in selective breeding to improve the reproductive traits of sheep.

[0050] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for rapidly detecting single nucleotide polymorphism of sheep INSL3 gene using PCR-SSCP, characterized in that: The whole genome DNA of the sheep to be tested is used as a template and the primer pair P is used as a primer to amplify the sheep INSL3 gene fragment by PCR, the PCR amplified fragment is denatured into single-stranded DNA, and then detected by polyacrylamide gel electrophoresis, and the genotype of the single nucleotide polymorphism site on the sheep INSL3 gene is determined according to the gel electrophoresis result; the single nucleotide polymorphism of the sheep INSL3 gene is the single nucleotide polymorphism of the 120th A>T mutation of the sheep INSL3 gene; The nucleotide sequence of the primer pair P is: Forward primer F: SEQ ID NO.1, Reverse primer R: SEQ ID NO.

2.

2. The method for rapid detection of single nucleotide polymorphism of sheep INSL3 gene using PCR-SSCP according to claim 1, characterized in that: The PCR amplification reaction system is: 0.5 μL DNA, 0.5 μL forward primer and 0.5 μL reverse primer, 7.5 μL PCR Mix and 6 μL ddH2O, with a total volume of 15 μL; the PCR amplification reaction program is: 95°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, for a total of 35 cycles.

3. The method for rapid detection of single nucleotide polymorphism of sheep INSL3 gene using PCR-SSCP according to claim 1, characterized in that: The polyacrylamide gel electrophoresis detection conditions are: 140V, 10min, followed by 120V, 10h.

4. The method for rapid detection of single nucleotide polymorphism of sheep INSL3 gene using PCR-SSCP according to claim 1, characterized in that: The single nucleotide polymorphism of the sheep INSL3 gene shows three genotypes: AA, AT and TT, among which AT is the dominant genotype, which is beneficial to improving the reproductive traits of sheep.

5. Application of the method for rapid detection of single nucleotide polymorphism of sheep INSL3 gene by PCR-SSCP as described in claims 1 to 4 in assisted selection and molecular breeding related to sheep reproductive traits.

6. The use according to claim 5, characterized in that: The sheep are selected from Xinjiang local Cele black sheep, Kazakh sheep and Duolang sheep.

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