A method and application of rapid detection of single nucleotide polymorphism at Chr1: g. 61542435 site in sheep using KASP

Abstract

The present invention discloses a method and application for rapidly detecting single nucleotide polymorphisms at the Chr1:g.61542435 site in sheep using KASP. Using sheep genomic DNA as a template and the published specific KASP primer sequence, the KASP detection method is used to rapidly and accurately type the single nucleotide polymorphisms at the Chr1:g.61542435 site in sheep. The different genotypes of the SNP site are significantly correlated with the milk protein rate trait of sheep milk, and play an important role in the molecular marker-assisted selection of milk protein rate in sheep milk. The present invention can assist in establishing a sheep genetic resource group with a high milk protein rate by rapidly and accurately detecting the mutant genotype of the Chr1:g.61542435 site related to the milk protein trait of sheep milk, thereby improving the quality of sheep milk products.

Description

Technical Field

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

[0002] Optimizing the milk protein ratio in sheep milk helps improve the composition of ewes' milk. High-quality milk, rich in immunoglobulins and other nutrients, helps boost lamb immunity and reduce the risk of disease. Furthermore, adequate nutrition helps lambs better adapt to the external environment, thereby increasing their survival rate. Furthermore, optimizing milk protein ratio is often closely linked to improved mammary gland development and lactation performance. Therefore, optimizing milk protein ratio helps ewes produce more milk, meeting the growth needs of their lambs.

[0003] Through genetic improvement, such as selecting breeding sheep with excellent milk protein content, the milk protein content of sheep milk can be gradually optimized, increasing the milk yield and composition of ewes. As an important economic trait, milk protein content has been extensively studied genetically in livestock such as sheep. For example, genes such as the β-lactoglobulin (BLG), α-lactalbumin (α-LA), and casein (CSN) genes have all been reported to potentially regulate traits such as milk protein content in sheep. Furthermore, the expression of these genes is also regulated by various mechanisms, including hormones and transcription factors.

[0004] Like other economic traits, the milk protein content trait in sheep is regulated by multiple, micro-effect genes, making it difficult to identify the major gene. However, by identifying molecular markers closely linked to milk protein content, marker-assisted selection (MAS) can be implemented, improving breeding efficiency. This patent discloses a KASP rapid detection method for the G>A variant at Chr1: g. 61542435 in sheep, and its application in molecular marker-assisted selection breeding for the milk protein content trait in sheep milk. Summary of the Invention

[0005] The present invention discloses a method and application of rapidly detecting single nucleotide polymorphisms at the Chr1: g. 61542435 site in sheep using KASP. The specific KASP primers disclosed in the present invention can rapidly and accurately detect mutations at the Chr1: g. 61542435 site G>A variant, which is associated with the milk protein rate trait in sheep milk. This can assist in establishing a sheep genetic resource population with a high milk protein rate, thereby improving the quality of sheep milk products.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

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

[0008] The specific KASP primers are characterized by the following sequence: the site G typing primer FAM, the site A typing primer VIC sequence and the universal primer sequence:

[0009] Site G typing primer FAM:

[0010] 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3':

[0011] Site A typing primer VIC:

[0012] 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3';

[0013] Universal primers:

[0014] 5'-TCAGGACTTCCCTTTCTACATTCG-3'.

[0015] The reaction program was as follows: pre-denaturation at 94°C for 15 min; 10 cycles of denaturation at 94°C for 20 s and annealing at 61°C for 1 min, with the annealing temperature decreasing by 0.6°C after each cycle; and 26 cycles of denaturation at 94°C for 20 s and annealing at 55°C for 1 min.

[0016] The method for rapidly detecting single nucleotide polymorphisms at the Chr1: g. 61542435 site in sheep using KASP and the genotype determination method thereof are characterized in that the three genotypes of GG, GA, and AA at the Chr1: g. 61542435 site are typed according to the intensities of different types of fluorescence that match the linker sequences carried by the KASP primers.

[0017] The present invention further discloses the application of a method for rapid detection of single nucleotide polymorphisms (SNPs) at the Chr1: g. 61542435 locus in sheep molecular marker-assisted selection breeding using KASP. The reference genome version for the Chr1: g. 61542435 locus is ARS-UI_Ramb_v3.0. The method is primarily used in molecular marker-assisted selection breeding for the milk protein yield trait in sheep milk. Experimental results demonstrate that the GG genotype, located at the G>A variant at Chr1: g. 61542435 in the sheep reference sequence ARS-UI_Ramb_v3.0, can serve as a molecular marker for improving the milk protein yield trait in sheep milk.

[0018] The method and application disclosed in the present invention for rapid detection of single nucleotide polymorphisms at the Chr1: g. 61542435 site in sheep using KASP have the following positive effects compared with the prior art:

[0019] (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 a site detection accuracy of >99.8%;

[0020] (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.

[0021] (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.

[0022] (4) For the first time, it was disclosed that the single nucleotide polymorphism mutation at the Chr1: g. 61542435 site in sheep is significantly associated with the milk protein rate trait in sheep milk, and it was pointed out that this site can be used in molecular marker-assisted selection breeding for the milk protein rate trait in sheep milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the KASP genotype map of sheep Chr1: g. 61542435;

[0024] Figure 2 Bar chart of association analysis between sheep milk protein rate and target SNP. DETAILED DESCRIPTION

[0025] The present invention is described below by way of specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments should be understood to be illustrative rather than limiting the scope of the present invention, the spirit and scope of the present invention being limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention. The raw materials and reagents used in the present invention are all commercially available. Example

[0026] 1. 136 Huyang reserve ewes (all from Tianjin Tengyuan Animal Husbandry Co., Ltd.) were used as test subjects. Through uniform breeding and the same postpartum feeding and management conditions, the somatic cell count characteristics of their sheep milk were measured and recorded.

[0027] 2. Collect 2 mL of anticoagulated blood from the jugular vein of all test sheep and extract the target individual DNA using the phenol-imide method;

[0028] 3. Specific KASP primers and universal primers were used. The specific sequence information is as follows:

[0029] Site G typing primer FAM:

[0030] 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3':

[0031] Site A typing primer VIC:

[0032] 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3';

[0033] Universal primers:

[0034] 5'-TCAGGACTTCCCTTTCTACATTCG-3'.

[0035] 4. Using the extracted DNA as a template, perform typing amplification using the specific KASP primers and universal primers from step 3. The amplification procedure is as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61°C for 1 minute, for a total of 10 cycles, with the annealing temperature lowered by 0.6°C after each cycle; denaturation at 94°C for 20 seconds, annealing at 55°C for 1 minute, for a total of 26 cycles. The final KASP typing pattern for the sheep Chr1: g. 61542435 locus is shown below. Figure 1As shown; the three genotypes of GG, GA, and AA at the Chr1: g. 61542435 site were typed according to the intensities of different types of fluorescence that matched the linker sequences carried by the KASP primers.

[0036] 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 locus was GG, the milk protein rate was 5.59±0.15%; when the genotype of Chr1: g. 61542435 locus was GA, the milk protein rate was 5.23±0.10%; when the genotype of Chr1: g. 61542435 locus 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).

[0037] 6. Based on the analysis of milk protein content in primiparous Hu sheep, blood can be collected from newly born Hu sheep, DNA extracted, and then the patented KASP rapid genotyping technology can be used to determine the milk protein content of the sheep after mating and giving birth. To select Hu sheep with high milk protein content, the offspring with the GG genotype at the Chr1: g. 61542435 locus should be continuously selected and retained, while the offspring with the AA genotype at the Chr1: g. 61542435 locus should be continuously eliminated.

Claims

1. A method for rapid detection of single nucleotide polymorphisms at the Chr1:g.61542435 site in sheep using KASP in marker-assisted selection breeding of sheep, wherein the method is used in marker-assisted selection breeding of the milk protein trait in sheep milk; the method comprises the following steps: Using sheep genomic DNA as a template, a partial fragment containing the G>A variant site at Chr1: g. 61542435 was amplified using specific KASP primers, and the genotype of the G>A variant site at Chr1: g. 61542435 was identified. The sheep reference genome version for Chr1: g. 61542435 was ARS-UI_Ramb_v3.

0. The specific KASP primer sequences were: Site G typing primer FAM: 5'-gaaggtgaccaagttcatgctAGGAATTAGAACTGGGAGAAGAGC-3': Site A typing primer VIC: 5'-gaaggtcggagtcaacggattAGGAATTAGAACTGGGAGAAGAGT-3'; Universal primers: 5'-TCAGGACTTCCCTTTCTACATTCG-3'; GG genotype is used as the genotype for improving the milk protein rate of sheep milk; the sheep are Hu sheep.

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