Sheep BMPR1B gene single nucleotide polymorphism detection primer and application

Abstract

The invention relates to a sheep BMPR1B gene single nucleotide polymorphism detection primer and application, and belongs to the technical field of genes. The SNP molecular marker corresponding to the primer is located on the sixth chromosome of the BMPR1B gene, and the primer is used for marker-assisted selective breeding of sheep. The invention provides a plurality of SNP molecular marker sites related to sheep breeding traits, and the genotype of each site is judged by a method of taking sheep blood genome DNA as a template and directly sequencing a PCR product. The molecular marker can be used as an effective molecular marker for improving the breeding traits of the sheep, the detection method is simple and rapid, the accuracy is high, the breeding efficiency of the sheep can be improved, the application value is very high, and more ways are provided for screening and researching the breeding traits of the sheep.

Description

Technical Field

[0001] The invention relates to a sheep BMPR1B gene single nucleotide polymorphism detection primer and application thereof, belonging to the field of gene technology. Background Art

[0002] Improved breeds are the foundation of modern animal husbandry production. Strengthening the protection and utilization of breed resources and improving the degree of improved breeds play an important role in promoting the high-quality development of animal husbandry. Huyang is an animal of the genus Ovis in the family Bovidae of the order Artiodactylus of the order Mammalia. It has the characteristics of "multiple births, estrus throughout the year, and suitable for shed feeding". It has been widely introduced throughout the country as the first maternal breed. Huyang has high fertility. Except for primiparous ewes, they generally produce more than 2 lambs per litter, and some can produce 4 to 6 lambs per litter. There are rich sheep breeds in my country, but there are few breeds with high fertility. Therefore, by strengthening the selection and breeding of Huyang to improve quality and expand the number of groups, it is possible to achieve the transformation of high-quality germplasm resources into advantageous economic benefits and promote the high-quality development of the mutton sheep industry. The BMPR1B gene is the earliest and most widely studied gene related to sheep reproductive traits, among which the FecB gene is the main effect gene for sheep multiple births. The FecB gene is located in the 8th exon of the BMPR1B gene on chromosome 6 of sheep and is located in the protein kinase functional domain. FecB gene mutations lead to missense mutations in the 746th base of the BMPR1B gene coding sequence, which can significantly increase the number of ovulations and lambing in sheep. The FecB gene was first discovered in the Booroola Merino sheep in Australia and was first proposed by Piper and Bindon in 1980. In 2009, Hua et al. summarized the population distribution and gene frequency of the FecB gene in some sheep breeds in China and found that the FecB gene is distributed in multiple high-fertility breeds, such as Hu sheep and small-tailed Han sheep. The BMPR1B gene is of great significance in the reproductive performance of sheep and is widely used to improve the reproductive effect of sheep. In recent years, researchers have been introducing the FecB gene into sheep flocks with low fertility through hybridization and other methods, in order to improve the reproductive performance of the group and thus increase economic benefits; although some BMPR1B gene loci have been disclosed for research on improving sheep reproduction rates, the small number of loci cannot meet breeding needs. Summary of the invention

[0003] The purpose of the present invention is to propose a single nucleotide polymorphism detection primer and application of the sheep BMPR1B gene in view of the defects of the prior art, and to provide a plurality of SNP molecular marker sites related to the reproductive traits of Hu sheep for screening research on the reproductive traits of Hu sheep. The SNP molecular marker related to the reproductive traits of Hu sheep is located on chromosome 6 of the BMPR1B gene and has a significant correlation with the number of lambs in sheep. The reproductive trait described in the present invention is the number of lambs, and the screening and / or breeding of Hu sheep reproductive traits is to use the above-mentioned BMPR1B gene molecular marker to identify the number of lambs trait of Hu sheep.

[0004] The present invention solves the technical problem through the following technical solutions:

[0005] Firstly, sheep BMPR1B gene single nucleotide polymorphism detection primers are provided. The primers contain 4 groups, each group consists of an upstream primer and a downstream primer, and the nucleotide sequences thereof are shown in SEQ ID NO: 1-SEQ ID NO: 8.

[0006] Secondly, the primers are used for sheep marker-assisted selection breeding. The primer pairs are mainly used to detect the genotypes at four loci of the sheep BMPR1B gene chromosome 6. The primers and product sequences correspond to four SNP molecular markers related to Hu sheep reproductive traits, as shown in Table 1:

[0007] Table 1

[0008]

[0009]

[0010] The detection method comprises the following steps,

[0011] The first step is to use sheep genomic DNA as a template to amplify a fragment containing four single nucleotide polymorphism sites of the sheep BMPR1B gene to obtain an amplified product;

[0012] The second step is to analyze the amplified products and determine the genotypes of the four sheep BMPR1B gene single nucleotide polymorphism sites.

[0013] In the above method, different genotypes of the BMPR1B gene have a significant effect on the number of lambs born in sheep. The genotype is determined by direct sequencing of the PCR product, and the BMPR1B genotype is determined based on the sequencing peak graph. This method can retain individuals with high fertility genotypes, eliminate individuals with low fertility genotypes, and increase the number of lambs born in sheep generation by generation.

[0014] Furthermore, the reaction system for PCR amplification in the first step is 40 μL, including 20 μL of 2×Taq Plus MasterMixⅡ; 1.6 μL of upstream and downstream primers; 2 μL of template DNA; 14.8 μL of deionized water; the reaction procedure for PCR amplification is pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min; and storage at 4°C after the completion of PCR.

[0015] The judgment criteria of the second step are to select the C / C genotype at 30030903bp, the C / C genotype at 30050621bp, the T / T genotype at 30116134bp and the A / A genotype at 30116760bp as molecular markers for improving the reproductive traits of Hu sheep.

[0016] The present invention further uses the primers in a kit for detecting single nucleotide polymorphism of sheep BMPR1B gene.

[0017] The present invention further provides probe sequences for detecting the genotype at sites 30030903bp, 30050621bp, 30116134bp, and 30116760bp of chromosome 6 of the sheep BMPR1B gene, as shown in SEQ ID NO: 9 to SEQ ID NO: 16, as shown in Table 2:

[0018] Table 2

[0019]

[0020]

[0021] The present invention has found an effective SNP molecular marker that affects the number of lambs produced in sheep in the BMPR1B gene. Sheep are screened using the molecular marker of the present invention. The genotype of the molecular marker can be detected by PCR amplification and product direct sequencing using a kit containing the above-mentioned SNP molecular marker primers. The method is simple, fast, highly accurate and inexpensive. Individuals with high fertility genotypes are retained and individuals with low fertility genotypes are eliminated, which can improve the reproductive performance of sheep and accelerate the breeding progress of sheep. It has high application value and provides more ways for the screening research of sheep reproductive traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scatter plot of the gene mass spectrometry sequencing of the BMPR1B g.30030903 site.

[0023] Figure 2 This is the agarose gel electrophoresis diagram of the PCR product of BMPR1B g.30030903 site.

[0024] Figure 3 This is the sequencing peak diagram of the PCR product of BMPR1B g.30030903 site.

[0025] Figure 4 This is a scatter plot of the gene mass spectrometry sequencing of the BMPR1B g.30050621 site.

[0026] Figure 5 This is the agarose gel electrophoresis diagram of the PCR product of BMPR1B g.30050621 site.

[0027] Figure 6 This is the sequencing peak diagram of the PCR product of BMPR1B g.30050621 site.

[0028] Figure 7 This is a scatter plot of the gene mass spectrometry sequencing of the BMPR1B g.30116134 site.

[0029] Figure 8 This is the agarose gel electrophoresis diagram of the PCR product of BMPR1B g.30116134 site.

[0030] Fig. 9 This is the sequencing peak diagram of the PCR product of BMPR1B g.30116134 site.

[0031] Fig.10 This is a scatter plot of the gene mass spectrometry sequencing of the BMPR1B g.30116760 site.

[0032] Fig.11 This is the agarose gel electrophoresis diagram of the PCR product of BMPR1B g.30116760 site.

[0033] Fig.12 This is the sequencing peak diagram of the PCR product of BMPR1B g.30116760 site. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and related drawings. However, it is worth noting that the implementation of the present invention is not limited to this, and relevant personnel can implement it in other different forms without violating the purpose and spirit of the present invention.

[0035] Example 1

[0036] This example is about the typing and identification of the sheep BMPR1B gene SNP molecular marker 1. Experimental animals and sample collection

[0037] The Hu sheep used in this experiment came from Jiangsu Qianbao Animal Husbandry (October 20, 2023, company manager Wang Daxiang, phone 13912501950), a total of 705. All Hu sheep were healthy and grew up in the same breeding conditions and environment. 10mL of blood from pregnant ewes was collected from the jugular vein, placed in an anticoagulant tube containing EDTA, and stored at -20℃. Phenotypic information such as the number of lambs born by ewes was collected for subsequent association analysis experiments.

[0038] 2. Main instruments

[0039] Pipette (Eppendorf), electronic balance (HENGJI), microwave oven (Galanz), refrigerator (Haier), handheld centrifuge (SCILOGEX, S1010E), vortexer (Dalong), digital constant temperature water bath (Changzhou Putian, HH-G2), high-speed refrigerated centrifuge (Eppendorf, 5424R), micro-spectrophotometer (NANODROP2000), PCR instrument (Applied Biosystems), electrophoresis instrument (Beijing Liuyi, DYY-6C), and fully automatic digital gel imaging system (Tanon, Tanon-4100).

[0040] 3. Main Reagents

[0041] TIANGEN blood genomic DNA extraction kit (centrifugal column type), 50×TAE (Solarbio), agarose, 2×Taq Plus Master MixⅡ (Dye Plus) (Vazyme, P213-03), 10000×TS-GelRed nucleic acid gel dye (TSINGKE, TSJ003), DL2000 Plus DNA Marker (Vazyme, MD102-02).

[0042] 4. Methods

[0043] 4.1 Extraction of genomic DNA from Hu sheep blood

[0044] Take the Hu sheep blood out of the -20℃ refrigerator two hours in advance and put it in a 4℃ refrigerator to thaw. After confirming that the blood is completely thawed, extract the DNA from the whole blood according to the instructions of the TIANGEN blood genomic DNA extraction kit. Use a micro-spectrophotometer to detect the DNA concentration and quality, where OD260 / 280 should be between 1.80 and 2.00, and OD260 / 230 should be between 1.80 and 2.20. Qualified DNA samples are stored in a -20℃ refrigerator.

[0045] Example 2

[0046] Statistical analysis of BMPR1B gene SNP site polymorphism and its relationship with Hu sheep reproductive traits

[0047] 1. Statistics of genetic parameters of BMPR1B gene population

[0048] According to the method of using the primers in Table 1 for Example 1, 705 Hu sheep were genotyped, and the population genetic parameters of the locus were calculated, including genotype frequency, allele frequency, homozygosity, heterozygosity, effective allele number, polymorphic information content and Hardy-Weinberg equilibrium P value. The probe sequences used for mass spectrometry sequencing are shown in Table 2. The results are shown in Figure 1-Figure 12 As shown, a total of 4 SNP loci with significant effects on lamb number were screened out. Figure 3 Table 3 shows that there are three genotypes of TT, CT and CC in Hu sheep at the BMPR1B g.30030903 locus.

[0049] Table 3 Genetic parameters of BMPR1B g.30030903 locus gene population

[0050]

[0051] Figure 6 Table 4 shows that there are three genotypes of TT, CT and CC in Hu sheep at the BMPR1B g.30050621 locus.

[0052] Table 4 Genetic parameters of BMPR1B g.30050621 locus gene population

[0053]

[0054] Fig. 9 As shown in Table 5, there are three genotypes of BMPR1B g.30116134 in Hu sheep: GG, GT, and TT.

[0055] Table 5 Genetic parameters of BMPR1B g.30116134 locus gene population

[0056]

[0057] Fig.12 As shown in Table 6, there are three genotypes of CC, CA and AA in Hu sheep at the BMPR1B g.30116760 locus.

[0058] Table 6 Genetic parameters of BMPR1B g.30116760 locus gene population

[0059]

[0060] 2. Association between BMPR1B gene SNP loci and Hu sheep reproductive traits

[0061] SAS (9.4) software was used to perform least squares statistical analysis using the GLM procedure to analyze the association between different genotypes of the BMPR1B gene locus and the reproductive traits of Hu sheep.

[0062] Taking genotype and season as fixed effects, the model is: Yijk = μ + Gi + Sj + eijk. In the formula, Yijk is the phenotypic value of individual lambing number; μ is the population mean; Gi is the genotype effect; Sj is the season effect; eijk is the random error.

[0063] 2.1 Analysis of association between BMPR1B gene SNP loci and Hu sheep reproductive traits

[0064] The fixed effects with no significant correlation with individual phenotypic values ​​other than genotype effects were deleted, and the association analysis between BMPR1B gene and the number of lambs born in Hu sheep was conducted. The results were expressed as "least square mean ± standard error".

[0065] The results are shown in Table 7. BMPR1B g.30030903 has a significant effect on the reproductive traits of Hu sheep.

[0066] Table 7 Results of association analysis between BMPR1B g.30030903 locus and Hu sheep reproductive traits

[0067]

[0068] The analysis results of the other three sites are shown in Tables 8 to 10.

[0069] Table 8 Results of association analysis between BMPR1B g.30050621 locus and Hu sheep reproductive traits

[0070]

[0071] Table 9 Results of association analysis between BMPR1B g.30116134 locus and Hu sheep reproductive traits

[0072]

[0073] Table 10 Association results between BMPR1B g.30116760 locus and Hu sheep reproductive traits

[0074]

[0075] This example provides the following research sites for comparison: the method is the same as that in Example 2 and will not be repeated here.

[0076] The following Tables 11 to 17 are the population genetic parameters of SNP sites that have no significant effect on the number of lambs:

[0077] Table 11

[0078]

[0079] Table 12

[0080]

[0081] Table 13

[0082]

[0083] Table 14

[0084]

[0085] Table 15

[0086]

[0087] Table 16

[0088]

[0089] Table 17

[0090]

[0091] Tables 18 to 24 below show the results of association analysis between SNP loci that have no significant effect on the number of lambs and the reproductive traits of Hu sheep:

[0092] Table 18 Results of association analysis between ACVR1 g.151895574 locus and Hu sheep reproductive traits

[0093]

[0094] Table 19 Results of association analysis between BMPR1B g.30196018 locus and Hu sheep reproductive traits

[0095]

[0096] Table 20 Results of association analysis between COL4A4 g.229401333 locus and Hu sheep reproductive traits

[0097]

[0098] Table 21 Results of association analysis between COL4A4 g.229307182 locus and Hu sheep reproductive traits

[0099]

[0100] Table 22 Results of association analysis between CYP11B1 g.14478149 locus and Hu sheep reproductive traits

[0101]

[0102] Table 23 Results of association analysis between RASGRF1 g.23015418 locus and Hu sheep reproductive traits

[0103]

[0104] Table 24 Results of association analysis between RASGRF1 g.23015977 locus and Hu sheep reproductive traits

[0105]

[0106] Example 3

[0107] Methods to improve the reproductive traits of Hu sheep

[0108] The method of improving the reproductive traits of Hu sheep mainly includes the following steps:

[0109] 1. Collect blood from the Hu sheep to be tested and extract blood genomic DNA.

[0110] 2. Using the primers described in Example 1, PCR amplification was performed with blood genomic DNA as a template, and the genotype was determined by direct sequencing of the PCR amplification product.

[0111] in:

[0112] (1) PCR amplification system (20 μL): 2× Taq Plus Master MixⅡ 10 μL; upstream and downstream primers 0.6 μL each; template DNA 1 μL; deionized water 7.8 μL.

[0113] (2) PCR amplification procedure: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 7 min; storage at 4°C after PCR.

[0114] (3) Agarose gel electrophoresis of PCR products: Prepare 2% agarose gel. After the agarose gel solidifies, add samples, including 9 μL of PCR product and 5 μL of DNA marker. Then put the agarose gel into an electrophoresis tank filled with 1×TAE (the 1×TAE liquid level must cover the gel surface) and run the gel at 140V for 35 minutes. After the electrophoresis is completed, transfer the gel to the imaging system for observation. The results are as follows: Figure 2 As shown, the product size was consistent with the expected specificity.

[0115] (4) Sequencing: The PCR product and primer P1 were sent to Qingke Biotechnology Co., Ltd. for direct sequencing.

[0116] (5) Genotype determination: SnapGene software was used to compare and analyze the sequencing peaks of BMPR1B g.30030903, BMPR1B g.30116760, BMPR1B g.30116134, and BMPR1B g.30050621. Figure 3 , Figure 6 , Fig. 9 and Fig.12 As shown, when the peak graph at the target position shows a single peak, the genotype is CC, AA, GG or TT; when the peak graph shows a double peak, the genotype is CT, GT or CA.

[0117] 3. The CC genotype of BMPR1B g.30030903, the AA genotype of BMPR1B g.30116760, the TT genotype of BMPR1Bg.30116134, and the CC genotype of BMPR1B g.30050621 can be used as effective molecular markers to improve the reproductive traits of Hu sheep.

[0118] Selecting individuals with CC genotype of BMPR1B g.30030903, AA genotype of BMPR1B g.30116760, TT genotype of BMPR1Bg.30116134, and CC genotype of BMPR1B g.30050621 as parents in Hu sheep breeding can improve the reproductive traits of offspring.

[0119] By using the SNP molecular markers of the present invention to screen Hu sheep, retaining individuals with high fertility genotypes for genetic selection and breeding, and eliminating individuals with low fertility genotypes, the breeding goal of improving the reproductive performance of Hu sheep can be achieved.

[0120] In addition to the above implementations, the present invention may also have other implementations. Any technical solution formed by equivalent replacement or equivalent transformation falls within the protection scope required by the present invention.

Claims

1. A single nucleotide polymorphism detection primer for sheep BMPR1B gene, characterized in that: The primers contain 4 groups, each group consists of an upstream primer and a downstream primer, and the nucleotide sequences thereof are shown in SEQ ID NO: 1-SEQ ID NO:

8.

2. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 1, characterized in that: The primers are used for marker-assisted selection breeding of lamb size in sheep.

3. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 2, characterized in that: The primer pairs are used to detect the genotypes at the 30030903bp, 30050621bp, 30116134bp and 30116760bp sites of chromosome 6 of the sheep BMPR1B gene, respectively.

4. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 3, characterized in that: The detection method comprises the following steps, The first step is to use sheep genomic DNA as a template to amplify a fragment containing four single nucleotide polymorphism sites of the sheep BMPR1B gene to obtain an amplified product; The second step is to analyze the amplified products and determine the genotypes of the four sheep BMPR1B gene single nucleotide polymorphism sites.

5. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 4, characterized in that: The reaction system of PCR amplification in the first step is 40 μL, including 20 μL of 2×Taq Plus Master MixⅡ; 1.6 μL of upstream and downstream primers; 2 μL of template DNA; 14.8 μL of deionized water; the reaction procedure of PCR amplification is 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing for 20 s, 72°C extension for 30 s, a total of 30 cycles; 72°C extension for 5 min; storage at 4°C after PCR.

6. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 5, characterized in that: The sequences of the amplified products are shown in SEQ ID NO: 17 to SEQ ID NO:

24.

7. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 4, characterized in that: The judgment standard of the second step is to select the C / C genotype at 30030903 bp, the C / C genotype at 30050621 bp, the T / T genotype at 30116134 bp and the A / A genotype at 30116760 bp as molecular markers for improving sheep reproductive traits.

8. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 2, characterized in that: The primers are used in a kit for detecting single nucleotide polymorphism of sheep BMPR1B gene.

9. The use of the sheep BMPR1B gene single nucleotide polymorphism detection primer according to claim 3, characterized in that: The probe sequences for detecting the genotypes at sites 30030903bp, 30050621bp, 30116134bp, and 30116760bp on chromosome 6 of the sheep BMPR1B gene are shown in SEQ ID NO: 9 to SEQ ID NO: 16.

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