A primer for detecting single nucleotide polymorphism of sheep BMPR1B gene and its application

Abstract

The present invention relates to a single nucleotide polymorphism detection primer for the sheep BMPR1B gene and its application, belonging to the field of gene technology. The SNP molecular marker corresponding to the primer of the present invention is located on chromosome 6 of the BMPR1B gene, and the primer is used for sheep marker-assisted selection breeding. The present invention provides a plurality of SNP molecular marker sites related to sheep reproductive traits, and determines the genotype of each site by directly sequencing the PCR product using sheep blood genomic DNA as a template. The present invention can be used as an effective molecular marker to improve sheep reproductive traits. The detection method is simple, rapid, and highly accurate, which can improve the breeding efficiency of sheep. It has high application value and provides more ways for the screening research of sheep reproductive traits.

Description

Technical Field

[0001] The present 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 livestock production. Strengthening the conservation and utilization of breed resources and improving the quality of improved breeds are crucial for promoting high-quality development of the livestock industry. The Hu sheep, a species of the genus Ovis in the family Bovidae, order Artiodactyla, class Mammalia, is characterized by high parity, year-round estrus, and suitability for confinement. It has been widely introduced throughout China as the primary maternal breed. Hu sheep have a high reproductive capacity. Except for primiparous ewes, they generally produce more than two lambs per litter, and some can produce four to six lambs per litter. my country has a rich variety of sheep breeds, but relatively few have high reproductive capacity. Therefore, strengthening the selection and breeding of Hu sheep to improve quality and expand the population can transform 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 extensively studied gene associated with reproductive traits in sheep, of which the FecB gene is the major gene for high parity in sheep. The FecB gene is located in exon 8 of the BMPR1B gene on chromosome 6 of sheep, within the protein kinase domain. Mutations in the FecB gene result in a missense mutation at base 746 of the BMPR1B coding sequence, which can significantly increase ovulation and lambing in sheep. The FecB gene was first discovered in 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 several Chinese sheep breeds and found that the FecB gene is present in several high-fertility breeds, such as the Hu sheep and the Small-tailed Han sheep. The BMPR1B gene plays a significant role in sheep reproductive performance and is widely used to improve sheep reproduction. 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 sites have been disclosed for research on improving sheep reproduction rates, due to the small number of sites, they cannot meet the needs of breeding. Summary of the Invention

[0003] The purpose of the present invention is to address the defects of the existing technology, propose a single nucleotide polymorphism detection primer for the sheep BMPR1B gene and its application, and provide multiple 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 sheep lambing. The reproductive trait described in the present invention is the number of lambs born, and the reproductive trait screening and / or breeding of Hu sheep is to use the above-mentioned BMPR1B gene molecular marker to identify the lamb number trait of Hu sheep.

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

[0005] First, 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 to SEQ ID NO: 8.

[0006] Secondly, the primers were used in sheep marker-assisted selection breeding. Primarily, the primer pairs were used to detect the genotypes at four loci on chromosome 6 of the sheep BMPR1B gene. The primer and product sequences corresponded to four SNP molecular markers associated with 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 BMPR1B genotypes significantly affect lambing size in sheep. Genotypes are determined by direct sequencing of PCR products, and BMPR1B genotypes are determined based on sequencing peaks. This method can retain individuals with high-fertility genotypes and eliminate those with low-fertility genotypes, thereby increasing lambing size over generations.

[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; and 14.8 μL of deionized water. The reaction procedure for the PCR amplification is pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 30 seconds, for a total of 30 cycles; extension at 72°C for 5 minutes; 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 the sheep BMPR1B gene on chromosome 6, 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 discovered an effective SNP molecular marker in the BMPR1B gene that affects lambing size in sheep. Using this molecular marker, sheep can be screened using a kit containing primers for the SNP molecular marker, and the genotype of the molecular marker can be detected through PCR amplification and direct product sequencing. This method is simple, rapid, highly accurate, and inexpensive. By retaining individuals with high-fertility genotypes and eliminating those with low-fertility genotypes, the method can improve sheep reproductive performance and accelerate sheep breeding progress. This method has high application value and provides more avenues for screening research on reproductive traits in sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a scatter plot of the 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 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 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] Figure 9 This is the sequencing peak diagram of the PCR product of BMPR1B g.30116134 site.

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

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

[0033] Figure 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 with reference to the embodiments and related drawings. However, it should be noted that the embodiments of the present invention are not limited thereto, and relevant personnel can implement the present invention 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 SNP molecular markers of sheep BMPR1B gene 1. Experimental animals and sample collection

[0037] The Hu sheep used in this study were 705 from Jiangsu Qianbao Animal Husbandry (October 20, 2023, company manager Wang Daxiang, phone 13912501950). All were healthy and raised under identical conditions and environments. 10 mL of blood was collected from the jugular vein of pregnant ewes, placed in anticoagulant tubes containing EDTA, and stored at -20°C. Phenotypic information, such as lambing number, was collected for subsequent association analysis.

[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 (spin 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] Two hours in advance, remove Hu sheep blood from a -20°C freezer and thaw in a 4°C refrigerator. Once the blood is completely thawed, extract DNA from the whole blood according to the instructions of the TIANGEN Blood Genomic DNA Extraction Kit. DNA concentration and quality are measured using a microspectrophotometer. OD260 / 280 should be between 1.80 and 2.00, and OD260 / 230 should be between 1.80 and 2.20. DNA samples that pass the test are stored in a -20°C refrigerator.

[0045] Example 2

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

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

[0048] 705 Hu sheep were genotyped using the primers in Table 1 as described in Example 1, and the population genetic parameters of the locus were calculated, including genotype frequency, allele frequency, homozygosity, heterozygosity, effective number of alleles, 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 Table 2. Figures 1-12 As shown in the figure, a total of 4 SNP sites with significant effects on lamb number were screened out. Figure 3 Table 3 shows that there are three genotypes of BMPR1B g.30030903 in Hu sheep: TT, CT, and CC.

[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 BMPR1B g.30050621 in Hu sheep: TT, CT, and CC.

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

[0053]

[0054] Figure 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] Figure 12 As shown in Table 6, there are three genotypes of CC, CA, and AA at the BMPR1B g.30116760 locus in Hu sheep.

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

[0059]

[0060] 2. Association between BMPR1B gene SNPs 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 individual phenotypic value of the lambing trait; μ is the population mean; Gi is the genotype effect; Sj is the season effect; and eijk is the random error.

[0063] 2.1 Association analysis between BMPR1B gene SNPs 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 between BMPR1B gene and lambing number of Hu sheep was analyzed. 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 Association analysis results 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 Association analysis results between BMPR1B g.30050621 locus and Hu sheep reproductive traits

[0070]

[0071] Table 9 Association analysis results 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 a study of the following sites for comparison: the method is the same as that in Example 2 and will not be repeated here.

[0076] Tables 11 to 17 below show the population genetic parameters of SNP sites that have no significant effect on lambing size:

[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 SNPs that have no significant effect on lambing number and 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 Association analysis results 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 Association analysis results between COL4A4 g.229307182 locus and Hu sheep reproductive traits

[0099]

[0100] Table 22 Association analysis results between CYP11B1 g.14478149 locus and Hu sheep reproductive traits

[0101]

[0102] Table 23 Association analysis results between RASGRF1 g.23015418 locus and Hu sheep reproductive traits

[0103]

[0104] Table 24 Association analysis results 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. PCR amplification was performed using the primers described in Example 1 and blood genomic DNA as a template, and the genotype was determined by direct sequencing of the PCR amplification products.

[0111] in:

[0112] (1) PCR amplification system (20 μL): 2× Taq Plus Master Mix II 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, 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 the sample, where the amount of PCR product added is 9 μL and the amount of DNA marker added is 5 μL. Then put the agarose gel into the electrophoresis tank filled with 1×TAE (the 1×TAE liquid level must cover the gel surface) and run the electrophoresis 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 size and had good specificity.

[0115] (4) Sequencing: The PCR product and primer P1 were sent to Qingke Biotechnology Company 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 、 Figure 9 and Figure 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, individuals with high reproductive capacity genotypes are retained for genetic selection and breeding, and individuals with low reproductive capacity genotypes are eliminated, the breeding goal of improving the reproductive performance of Hu sheep can be achieved.

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

Claims

1. Application of a single nucleotide polymorphism detection primer for the BMPR1B gene of Hu sheep in marker-assisted selection breeding of Hu sheep litter size, 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; the primers are respectively used to detect the genotypes of Hu sheep BMPR1B gene chromosome 6, wherein the 30030903bp corresponding sequence SEQ ID NO: 17 has a polymorphism of C / T at the 96th position from 5', the 30116760bp corresponding sequence SEQ ID NO: 19 has a polymorphism of C / A at the 268th position from 5', the 30116134bp corresponding sequence SEQ ID NO: 21 has a polymorphism of G / T at the 71st position from 5', and the 30050621bp corresponding sequence SEQ ID NO: 23 has a polymorphism of C / T at the 66th position from 5'.

2. The use of the single nucleotide polymorphism detection primers for the BMPR1B gene of Hu sheep according to claim 1 in marker-assisted selection breeding of Hu sheep litter size, characterized in that: The detection method comprises the following steps, The first step is to use Hu sheep genomic DNA as a template to amplify a fragment containing four Hu sheep BMPR1B gene single nucleotide polymorphism sites to obtain an amplified product; The second step is to analyze the amplified products and determine the genotypes of the four single nucleotide polymorphism sites of the Hu sheep BMPR1B gene.

3. The use of the single nucleotide polymorphism detection primers for the BMPR1B gene of Hu sheep according to claim 2 in marker-assisted selection breeding of Hu sheep litter size, characterized in that: The reaction system for PCR amplification in the first step is 40 μL, including 20 μL of 2×TaqPlus Master MixⅡ, 1.6 μL of upstream and downstream primers, 2 μL of template DNA, and 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, and 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.

4. The use of the single nucleotide polymorphism detection primers for the BMPR1B gene of Hu sheep according to claim 3 in marker-assisted selection breeding of Hu sheep litter size, characterized in that: The sequences of the amplified products are shown in SEQ ID NO: 17 to SEQ ID NO:

24.

5. The use of the single nucleotide polymorphism detection primers for the BMPR1B gene of Hu sheep according to claim 2 in marker-assisted selection breeding of Hu sheep litter size, characterized in that: The judgment criteria of the second step is 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 lambing number trait of Hu sheep.

6. Use of the single nucleotide polymorphism detection primers for the BMPR1B gene of Hu sheep according to claim 1 in marker-assisted selection breeding of Hu sheep litter size, characterized in that: The probe sequences for mass spectrometry sequencing for detecting the genotype at sites 30030903bp, 30050621bp, 30116134bp, and 30116760bp on chromosome 6 of the Hu sheep BMPR1B gene are shown in SEQ ID NO: 9 to SEQ ID NO: 16.

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