Method for rapidly detecting sheep Chr1: g.61541386 site single nucleotide polymorphism by using KASP and application

Abstract

The invention discloses a method for rapidly detecting the single nucleotide polymorphism of a sheep Chr1: g.61541386 site by using KASP (Kinase Assay Specific Protein) and application of the single nucleotide polymorphism of the sheep Chr1: g.61541386 site. According to the present invention, sheep genome DNA is adopted as a template, rapid and accurate typing is performed on the single nucleotide polymorphism of the sheep Chr1: g.61541386 site 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 body cell number character, such that the important effect is provided in the sheep anti-mastitis early stage selection; by rapidly and accurately detecting the Chr1: g.61541386 site mutation genotype related to the sheep milk cell number character, establishment of an anti-mastitis sheep genetic resource group can be assisted, so that the lactation performance of sheep offspring is improved.

Description

Technical Field

[0001] The present invention relates to a method for rapidly detecting single nucleotide polymorphism at Chr1: g. 61541386 site of sheep using KASP and its application, belonging to the field of sheep SNP molecular marker detection and application. Background Art

[0002] The somatic cell count in sheep milk usually includes white blood cells (such as neutrophils, macrophages, lymphocytes), red blood cells and epithelial cells. Under normal circumstances, the milk fluid cell count in healthy sheep mammary glands is at a low level. However, when the sheep mammary gland is infected or inflamed, the somatic cell count will increase significantly. When sheep have mastitis, the mammary tissue will undergo an inflammatory response, causing a large number of white blood cells (especially neutrophils) to flow into the milk to fight the infection. Therefore, the somatic cell count in the milk of sheep with mastitis will increase significantly, exceeding the normal range.

[0003] Mastitis can damage mammary tissue and affect lactation function, thus leading to a decrease in milk production. Severe mastitis may even cause permanent damage to mammary tissue, causing sheep to lose their ability to lactate. In addition, a high somatic cell count in sheep milk will affect the sensory quality and nutritional value of the milk. For example, milk with a high somatic cell count may have a strange smell, change color, or go bad. Through genetic selection, sheep groups with excellent milk production traits and mastitis resistance can be selected, which can reduce the somatic cell count of sheep milk and improve the milk production performance and disease resistance of sheep from the source.

[0004] The regulation of sheep milk somatic cell count may involve complex signaling pathways and regulatory networks. These pathways and networks may include signaling pathways related to immune response (such as the Jak-Stat signaling pathway), regulatory mechanisms related to mammary gland development and lactation, etc. Abnormalities in these pathways and networks may lead to mammary tissue dysfunction and aggravated inflammatory response, thereby affecting changes in milk somatic cell count. Earlier, it was found that a SNP molecular marker located in the intron of the sheep STAT5a gene was significantly correlated with the milk somatic cell count trait. Since the inheritance and regulation of sheep milk somatic cell count involve multiple micro-effect regulatory genes; therefore, there may be multiple SNP site association sites at the sheep genome level. This patent discloses a KASP rapid detection method for sheep Chr1: g. 61541386 site variation and its application in molecular marker-assisted selection breeding in sheep milk somatic cell traits. 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. 61541386 site in sheep. By using the specific KASP primers disclosed in the present invention, the mutation of the A>G variant site at Chr1: g. 61541386 site related to the somatic cell number trait of sheep milk can be quickly and accurately detected, and an anti-mastitis sheep genetic resource population is established, thereby improving the lactation performance of sheep offspring.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions A method and application of using KASP to quickly detect single nucleotide polymorphism at the Chr1: g. 61541386 site in sheep, characterized in that sheep genomic DNA is used as a template, a partial fragment containing the A>G variant site of Chr1: g.61541386 is amplified by specific KASP primers, and then the genotype of the A>G variant site of Chr1: g. 61541386 is identified, wherein the sheep reference genome version of the Chr1: g. 61541386 site is ARS-UI_Ramb_v3.0.

[0007] The specific KASP primers are characterized in that the site A typing primer FAM, the site G typing primer VIC sequence and the universal primer sequence are as follows: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3'; Universal Primer: 5'-CCATTTCCTTCTCCAATGCATGAA-3'.

[0008] The reaction procedure was as follows: 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 three genotypes of Chr1: g. 61541386, AA, AG, and GG, were typed according to the intensity of different types of fluorescence that matched the adapter sequence carried by the KASP primer.

[0009] The present invention further discloses the application of a method for rapidly detecting single nucleotide polymorphism at Chr1: g. 61541386 site of sheep using KASP in sheep molecular marker-assisted selection breeding, wherein the reference genome version of the Chr1: g. 61541386 site is ARS-UI_Ramb_v3.0. In particular, the application in molecular marker-assisted selection breeding for early selection of low somatic cell count and anti-mastitis traits in sheep milk. The AA genotype located at the A>G variant site at Chr1: g. 61541386 site of the sheep reference sequence ARS-UI_Ramb_v3.0 can be used as a molecular marker for improving sheep anti-mastitis and reducing sheep milk somatic cell count traits.

[0010] The method and application of the present invention for rapid detection of single nucleotide polymorphism at Chr1: g. 61541386 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. 61541386 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. 61541386 site in sheep. It has lower costs and only requires the cost of primer synthesis. It does not require the high cost of site-labeling probes and has a lower requirement for the amount of DNA sample.

[0011] (3) The KASP method was used to detect the single nucleotide polymorphism mutation at the Chr1: g. 61541386 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.

[0012] (4) For the first time, it was disclosed that the single nucleotide polymorphism mutation at the Chr1: g. 61541386 site in sheep is significantly associated with the sheep milk somatic cell count trait, and it was pointed out that this site can be used in the molecular marker-assisted selection breeding of sheep milk somatic cell count and anti-mastitis traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the KASP genotype map of sheep Chr1: g. 61541386 locus; Figure 2 Bar chart of association analysis between sheep milk somatic cell count and target SNP. DETAILED DESCRIPTION

[0014] 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

[0015] 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.

[0016] 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 A typing primer FAM: 5'-gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3'; Universal Primer: 5'-CCATTTCCTTCTCCAATGCATGAA-3'.

[0017] 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 as follows: 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. 61541386 site KASP typing diagram is shown below. Figure 1 As shown; the three genotypes of AA, AG, and GG at the Chr1: g. 61541386 site were typed according to the intensity of different types of fluorescence matching the linker sequence carried by the KASP primer.

[0018] 5. The results of the measurement of milk somatic cell count of primiparous Hu sheep ewes (mean ± standard error) showed that: when the genotype of Chr1: g. 61541386 was AA, the milk somatic cell count was 387.52±75.09 k / mL; when the genotype of Chr1: g. 61541386 was AG, the milk somatic cell count was 639.64±162.10 k / mL; when the genotype of Chr1: g. 61541386 was GG, the milk somatic cell count was 916.93±263.23 k / mL ( Figure 2 ); The number of milk somatic cells of AA genotype was significantly lower than that of GG genotype (P<0.05).

[0019] 6. According to the analysis results of the milk somatic cell count trait of primiparous Hu sheep, the high and low milk somatic cell count indicators of the newly born Hu sheep can be determined by blood sampling and DNA extraction, and then by the patented KASP rapid genotyping technology. In order to breed a group of Hu sheep with resistance to mastitis and low milk somatic cell count, the AA genotype offspring at the Chr1: g.61541386 locus in the flock should be continuously selected and retained, and the GG genotype offspring at the Chr1: g. 61541386 locus in the flock should be continuously eliminated.

Claims

1. A method for rapidly detecting single nucleotide polymorphism at Chr1: g. 61541386 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 A>G variant site of Chr1: g. 61541386 was amplified by specific KASP primers, and then the genotype of the A>G variant site of Chr1: g. 61541386 was identified, where the sheep reference genome version of Chr1: g.61541386 was ARS-UI_Ramb_v3.0; the specific KASP primer sequence was: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3'; Universal Primer: 5'-CCATTTCCTTCTCCAATGCATGAA-3'.

2. The method and application of rapid detection of single nucleotide polymorphism at Chr1: g.61541386 site in sheep using KASP as described in claim 1 is characterized by: The identification of the genotype specifically includes the following steps: typing the three genotypes of AA, AG, and GG at the Chr1: g. 61541386 site according to the intensity of different types of fluorescence that match the linker sequence carried by the KASP primer.

3. Application of the method for rapid detection of single nucleotide polymorphism at Chr1: g.61541386 site in 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 for early selection of low somatic cell count and anti-mastitis traits in sheep milk.

4. The use according to claim 3, characterized in that: The AA genotype located at the A>G variant site of the sheep reference sequence ARS-UI_Ramb_v3.0, Chr1: g. 61541386 site is used as a molecular marker for improving sheep's resistance to mastitis and reducing sheep milk somatic cell count traits.

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