Method for rapidly detecting single nucleotide polymorphism of sheep Chr1: g.61542435 site by using KASP and application

Abstract

The invention discloses a method for rapidly detecting the single nucleotide polymorphism of a sheep Chr1: g.61542435 site by using KASP (Kinase Assay Specific Protein) and application of the single nucleotide polymorphism of the sheep Chr1: g.61542435 site. According to the present invention, sheep genome DNA is adopted as a template, the single nucleotide polymorphism of the sheep Chr1: g.61542435 site is subjected to rapid and accurate typing through the published specific KASP primer sequence by using the KASP detection method, and the different genotypes of the SNP site are significantly related to the sheep milk protein rate character, such that the important effect is provided in the sheep milk protein rate molecular marker-assisted selection; according to the present invention, the Chr1: g.61542435 site mutation genotype related to the sheep milk protein character can be rapidly and accurately detected, such that the establishment of the sheep genetic resource group with the high milk protein rate can be assisted so as to improve the quality of the sheep milk product.

Description

Technical Field

[0001] The present invention relates to a method for rapidly detecting single nucleotide polymorphism at Chr1: g. 61542435 site of sheep using KASP and its application, belonging to the field of sheep SNP molecular marker detection and application. Background Art

[0002] Optimizing the milk protein rate trait of sheep milk helps to improve the milk composition of ewes. High-quality milk is rich in immunoglobulins and other nutrients, which helps to enhance the immunity of lambs and reduce the occurrence of diseases. At the same time, adequate nutrition supply also helps lambs to better adapt to the external environment, thereby improving their survival rate. In addition, the optimization of milk protein rate traits is usually closely related to the improvement of mammary gland development and lactation performance. Therefore, optimizing the milk protein rate trait helps ewes secrete more milk to meet the growth needs of lambs.

[0003] Through genetic improvement methods, such as selecting breeding sheep with excellent milk protein rate traits for breeding, the milk protein rate trait of sheep milk can be gradually optimized, and the milk production and milk composition of ewes can be improved. As an important economic trait, the milk protein rate trait has been widely studied genetically in livestock such as sheep. For example, the β-lactoglobulin (BLG) gene, α-lactalbumin (α-LA) gene, and casein (CSN) gene have all been reported to regulate traits such as sheep milk protein rate. At the same time, the expression of these genes is also regulated by a variety of regulatory mechanisms, including hormone regulation and transcription factor regulation.

[0004] The sheep milk protein rate trait, like other economic traits, is regulated by multiple micro-effect polygenes, and it is difficult to determine the major effect gene; however, by identifying molecular markers closely linked to the milk protein rate trait, molecular marker-assisted selection can be achieved to improve breeding efficiency. This patent discloses a KASP rapid detection method for the G>A variation at the Chr1: g. 61542435 site of sheep and its application in molecular marker-assisted selection breeding for the sheep milk protein rate trait. Summary of the invention

[0005] The present invention discloses a method and application of using KASP to quickly detect single nucleotide polymorphism at Chr1: g. 61542435 site of sheep. Through the specific KASP primers disclosed in the present invention, the mutation of the Chr1: g. 61542435 site G>A variant site related to the milk protein rate trait of sheep milk can be quickly and accurately detected, which can assist in establishing a sheep genetic resource group with a high milk protein rate, thereby improving the quality of sheep milk products.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A method and application of rapidly detecting single nucleotide polymorphism at Chr1: g. 61542435 site in sheep using KASP, characterized in that sheep genomic DNA is used as a template, a partial fragment containing the G>A variant site of Chr1: g.61542435 is amplified by specific KASP primers, and then the genotype of the G>A variant site of Chr1: g. 61542435 is identified, wherein the sheep reference genome version of the Chr1: g. 61542435 site is ARS-UI_Ramb_v3.0.

[0008] The specific KASP primers are characterized in that the site G typing primer FAM, the site A typing primer VIC sequence and the universal primer sequence are as follows: Site G typing primer FAM: 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3': Site A typing primer VIC: 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3'; Universal Primer: 5'-TCAGGACTTCCCTTTCTACATTCG-3'.

[0009] The reaction program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing at 61°C for 1 min for a total of 10 cycles, with the annealing temperature decreasing by 0.6°C after each cycle; denaturation at 94°C for 20 s, annealing at 55°C for 1 min for a total of 26 cycles.

[0010] The method for rapidly detecting single nucleotide polymorphism at Chr1: g. 61542435 site of sheep using KASP and the genotype determination used therein are characterized in that three genotypes of GG, GA and AA at Chr1: g. 61542435 site are typed according to the intensity of different types of fluorescence matching the linker sequence carried by the KASP primer.

[0011] The present invention further discloses the application of a method for rapidly detecting single nucleotide polymorphisms at the Chr1: g. 61542435 site of sheep using KASP in sheep molecular marker-assisted selection breeding, wherein the reference genome version of the Chr1: g. 61542435 site is ARS-UI_Ramb_v3.0. The application is mainly in the molecular marker-assisted selection breeding of the milk protein rate trait of sheep milk. The experimental results show that the GG genotype located at the G>A variant site of the Chr1: g. 61542435 site of the sheep reference sequence ARS-UI_Ramb_v3.0 can be used as a molecular marker for improving the milk protein rate trait of sheep milk.

[0012] The method and application of the present invention for rapid detection of single nucleotide polymorphism at Chr1: g. 61542435 site in sheep using KASP have the following positive effects compared with the prior art: (1) The KASP method was used to detect the single nucleotide polymorphism mutation at the Chr1: g. 61542435 site in sheep, which has a more efficient detection accuracy and the site detection accuracy is >99.8%; (2) The KASP method is used to detect the single nucleotide polymorphism mutation at the Chr1: g. 61542435 site in sheep. It has lower costs and only requires primer synthesis. It does not require the high cost of site-labeled probes and has a lower requirement for the amount of DNA sample.

[0013] (3) The KASP method was used to detect the single nucleotide polymorphism mutation at the Chr1: g. 61542435 site in sheep. It has strong detection flexibility and can be detected together with multiple other mutation sites, meeting the needs of low, medium and high throughput individual detection.

[0014] (4) For the first time, it was disclosed that the single nucleotide polymorphism mutation at the Chr1: g. 61542435 site of sheep is significantly associated with the milk protein rate trait of sheep milk, and it was pointed out that this site can be used in the molecular marker-assisted selection breeding of the milk protein rate trait of sheep milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the KASP genotype map of sheep Chr1: g. 61542435 locus; Figure 2 Bar chart of association analysis between sheep milk protein rate and target SNP. DETAILED DESCRIPTION

[0016] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available. Example

[0017] 1. A total of 136 Huyang reserve ewes were used as test subjects (all from Tianjin Tengyuan Animal Husbandry Co., Ltd.). Through uniform breeding and the same postpartum feeding management conditions, the somatic cell count characteristics of their sheep milk were measured and recorded.

[0018] 2. Collect 2 mL of anticoagulated blood from the jugular vein of all test sheep and extract the target individual DNA using the phenol imitation method; 3. Use specific KASP primers and universal primers. The specific sequence information is as follows: Site G typing primer FAM: 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3': Site A typing primer VIC: 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3'; Universal Primer: 5'-TCAGGACTTCCCTTTCTACATTCG-3'.

[0019] 4. Using the extracted DNA as a template, the specific KASP primers and universal primers in step 3 were used for typing amplification detection. The amplification program was: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃ annealing for 1 min for a total of 10 cycles, with the annealing temperature reduced by 0.6℃ after each cycle; 94℃ denaturation for 20 s, 55℃ annealing for 1 min, for a total of 26 cycles. The final sheep Chr1: g. 61542435 site KASP typing diagram is shown below. Figure 1 As shown; the three genotypes of GG, GA, and AA at the Chr1: g. 61542435 site were typed according to the intensity of different types of fluorescence matching the linker sequence carried by the KASP primer.

[0020] 5. The results of the measurement of milk somatic cell count traits of primiparous Hu sheep ewes (mean ± standard error) showed that: when the genotype of Chr1: g. 61542435 was GG, the milk protein rate was 5.59±0.15%; when the genotype of Chr1: g. 61542435 was GA, the milk protein rate was 5.23±0.10%; when the genotype of Chr1: g. 61542435 was AA, the milk protein rate was 4.92±0.07% ( Figure 2 The milk protein rate of GG genotype was significantly higher than that of GA and AA genotypes (P<0.05).

[0021] 6. According to the analysis results of the milk protein rate of primiparous Hu sheep, the milk protein rate of newly born Hu sheep can be determined by blood sampling and DNA extraction, and then the patented KASP rapid genotyping technology can be used to determine the milk protein rate of adult breeding and production. In order to breed Hu sheep with high milk protein rate, the offspring with GG genotype at the Chr1: g. 61542435 site in the flock should be continuously selected and retained, and the offspring with AA genotype at the Chr1: g. 61542435 site in the flock should be continuously eliminated.

Claims

1. A method for rapidly detecting single nucleotide polymorphism at Chr1: g.61542435 site in sheep using KASP, characterized in that: The following steps are involved: Using sheep genomic DNA as a template, a partial fragment containing the G>A variant site of Chr1: g. 61542435 was amplified by specific KASP primers, and then the genotype of the G>A variant site of Chr1: g. 61542435 was identified, wherein the sheep reference genome version of Chr1: g.61542435 site was ARS-UI_Ramb_v3.0; the sequence of the specific KASP primer was: Site G typing primer FAM: 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3': Site A typing primer VIC: 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3'; Universal Primer: 5'-TCAGGACTTCCCTTTCTACATTCG-3'.

2. The method for rapidly detecting single nucleotide polymorphism at Chr1: g.61542435 site in sheep using KASP according to claim 1, characterized in that: The identification of the genotype specifically includes the following steps: typing the three genotypes of GG, GA, and AA at the Chr1: g. 61542435 site according to the intensity of different types of fluorescence that match the linker sequence carried by the KASP primer.

3. The application of the method for rapid detection of single nucleotide polymorphism at Chr1: g.61542435 site of sheep using KASP as described in claim 1 in sheep molecular marker-assisted selection breeding, wherein the application refers to molecular marker-assisted selection breeding of sheep milk protein rate trait.

4. The use according to claim 3, characterized in that: The GG genotype located at the G>A mutation site at Chr1: g. 61542435 site in the sheep reference sequence ARS-UI_Ramb_v3.0 is used as a molecular marker for improving the milk protein rate trait of sheep milk.

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