A method for rapidly detecting single nucleotide polymorphism of sheep Chr1: g. 61541386 site by KASP and application thereof

Abstract

The application discloses a method for rapidly detecting single nucleotide polymorphism of a sheep Chr1:g.61541386 site by using KASP and application thereof. With sheep genomic DNA as a template, by using a specific KASP primer sequence published, the KASP detection method is used to rapidly and accurately type the single nucleotide polymorphism of the sheep Chr1:g.61541386 site, and different genotypes of the SNP site are significantly related to the milk somatic cell count trait of sheep and play an important role in early selection of sheep against mastitis. By rapidly and accurately detecting the Chr1:g.61541386 site mutation genotype related to the milk somatic cell count trait of sheep, the application can assist in establishing a genetic resource population of sheep against mastitis, so that the lactation performance of sheep offspring is improved.

Description

Technical Field

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

[0002] The somatic cell count in sheep milk typically includes white blood cells (such as neutrophils, macrophages, and lymphocytes), red blood cells, and epithelial cells. Under normal circumstances, the somatic cell count in the milk fluid of healthy sheep's mammary glands is low. However, when the mammary glands of sheep become infected or inflamed, the somatic cell count can increase significantly. When sheep suffer from mastitis, the mammary gland undergoes an inflammatory response, leading to a large influx of white blood cells (especially neutrophils) into the milk to combat the infection. Consequently, the somatic cell count in the milk of sheep with mastitis can be significantly elevated, exceeding the normal range.

[0003] Mastitis can damage mammary gland tissue, impairing lactation and leading to decreased milk production. Severe mastitis can even permanently damage mammary gland tissue, rendering the sheep unable to produce milk. Furthermore, a high somatic cell count in sheep milk can affect its sensory quality and nutritional value. For example, milk with a high somatic cell count may exhibit an off-flavor, discolor, or spoil. Genetic selection can be used to select sheep with superior milk production traits and resistance to mastitis, thereby reducing the somatic cell count in sheep milk and improving milk production and disease resistance.

[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 responses (such as the Jak-Stat signaling pathway) and regulatory mechanisms related to mammary gland development and lactation. Abnormalities in these pathways and networks may lead to mammary tissue dysfunction and increased inflammatory responses, thereby affecting changes in milk somatic cell count. Previously, a single-nucleotide polymorphism (SNP) molecular marker located within the intron of the sheep STAT5a gene was found to be significantly correlated with milk somatic cell count. Because the inheritance and regulation of sheep milk somatic cell count involve multiple micro-regulatory genes, multiple SNP-associated loci may exist within the sheep genome. This patent discloses a KASP rapid detection method for the sheep Chr1: g. 61541386 locus variation and its application in molecular marker-assisted selection breeding for sheep milk somatic cell traits. Summary of the Invention

[0005] The application discloses a method for rapidly detecting single nucleotide polymorphism of a Chr1: g. 61541386 site of sheep by KASP and application, and the method can rapidly and accurately detect mutation of an A>G variation site of the Chr1: g. 61541386 site related to a milk somatic cell count trait of sheep, and establishes a genetic resource population of sheep resistant to mastitis, so that the lactation performance of offspring of the sheep is improved.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions

[0007] A method for rapidly detecting single nucleotide polymorphism of a Chr1: g. 61541386 site of sheep by KASP and application, characterized by using sheep genomic DNA as a template, amplifying a partial fragment containing an A>G variation site of the Chr1: g. 61541386 site by specific KASP primers, and identifying the A>G variation site genotype of the Chr1: g. 61541386 site, wherein the sheep reference genome version of the Chr1: g. 61541386 site is ARS-UI_Ramb_v3.0.

[0008] The specific KASP primer is characterized by the following sequences of a site A typing primer FAM, a site G typing primer VIC and a universal primer:

[0009] The site A typing primer FAM is:

[0010] 5'-gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3';

[0011] The site G typing primer VIC is:

[0012] 5'-gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3';

[0013] The universal primer is:

[0014] 5'-CCATTTCCTTCTCCAATGCATGAA-3'.

[0015] The reaction procedure is as follows: 94 DEG C pre-denaturation for 15 min; 94 DEG C denaturation for 20 s, 61 DEG C annealing for 1 min for a total of 10 cycles, and the annealing temperature is reduced by 0.6 DEG C after each cycle; 94 DEG C denaturation for 20 s, 55 DEG C annealing for 1 min for a total of 26 cycles. The AA, AG and GG genotypes of the Chr1: g. 61541386 site are typed according to the intensity of different types of fluorescence matched with the linker sequence carried by the KASP primer.

[0016] The application further discloses application of the method for rapidly detecting the single nucleotide polymorphism of the sheep Chr1: g. 61541386 site by KASP in sheep 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 is applied in early selection of the molecular marker-assisted selection breeding of the sheep milk low somatic cell count and mastitis resistance traits. The AA genotype of the A>G variation site of the sheep reference sequence ARS-UI_Ramb_v3.0, Chr1: g. 61541386 site can be used as a molecular marker for improving the mastitis resistance of sheep and reducing the somatic cell count of sheep milk traits.

[0017] The method for rapidly detecting the single nucleotide polymorphism of the sheep Chr1: g. 61541386 site by KASP and the application disclosed by the application have the following advantages compared with the prior art:

[0018] (1) The KASP method is used to detect the single nucleotide polymorphism mutation of the sheep Chr1: g. 61541386 site, and the detection accuracy is higher than 99.8%.

[0019] (2) The KASP method is used to detect the single nucleotide polymorphism mutation of the sheep Chr1: g. 61541386 site, and the cost is lower, only the primer synthesis cost is needed, the high-cost site marker probe cost is not needed, and the requirement for the DNA sample amount is lower.

[0020] (3) The KASP method is used to detect the single nucleotide polymorphism mutation of the sheep Chr1: g. 61541386 site, and the detection flexibility is strong, the site can be detected together with multiple other mutation sites, and the low, medium and high throughput individual detection requirements can be met.

[0021] (4) The single nucleotide polymorphism mutation of the sheep Chr1: g. 61541386 site is disclosed for the first time, and the single nucleotide polymorphism mutation is significantly related to the somatic cell count of sheep milk, and the site can be used in the molecular marker-assisted selection breeding of the somatic cell count of sheep milk and mastitis resistance traits. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A KASP genotyping diagram of the sheep Chr1: g. 61541386 site;

[0023] Figure 2 A column chart of association analysis of the somatic cell count of sheep milk and the target SNP. DETAILED DESCRIPTION

[0024] The application will be described in the following with specific embodiments. Unless otherwise specified, the technical means used in the application are the methods well known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the application, the essence and scope of the application are only limited by the claims. For those skilled in the art, various changes or modifications to the ingredients and amounts of the materials in these embodiments without departing from the essence and scope of the application also belong to the protection scope of the application. The raw materials and reagents used in the application are commercially available. Examples

[0025] 1. 136 Hu sheep reserve ewes were used as test objects (all from Tianjin Tengyuan Livestock Breeding Co., Ltd.), and the number of somatic cells in sheep milk was determined under the conditions of uniform mating and the same postpartum feeding management.

[0026] 2. Collect 2 mL of anticoagulated blood from the jugular vein of all test sheep, and extract the target individual DNA using the phenol method;

[0027] 3. Specific KASP primers and universal primers were used, and the specific sequence information was as follows:

[0028] Site A typing primer FAM:

[0029] 5'- gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3';

[0030] Site G typing primer VIC:

[0031] 5'- gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3';

[0032] Universal primer:

[0033] 5'- CCATTTCCTTCTCCAATGCATGAA-3'.

[0034] 4. The extracted DNA was used as a template, and specific KASP primers and universal primers in step 3 were used for typing amplification detection, and 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, and the annealing temperature was reduced by 0.6℃ after each cycle; 94℃ denaturation for 20 s, 55℃ annealing for 1 min, a total of 26 cycles. The final obtained sheep Chr1: g. 61541386 site KASP typing map is as follows: Figure 1The three genotypes of Chr1: g. 61541386 site were genotyped according to the intensity of different types of fluorescence matched with the sequence of the adapter carried by the KASP primer.

[0035] 5. The statistical determination results (mean ± standard error) of the milk somatic cell count trait of the first-litter Hu ewes showed that: when the genotype of Chr1: g. 61541386 site was AA, the milk somatic cell count was 387.52 ± 75.09 k / mL; when the genotype of Chr1: g. 61541386 site was AG, the milk somatic cell count was 639.64 ± 162.10 k / mL; and when the genotype of Chr1: g. 61541386 site was GG, the milk somatic cell count was 916.93 ± 263.23 k / mL. Figure 2 The milk somatic cell count of AA genotype was significantly lower than that of GG genotype (P < 0.05).

[0036] 6. According to the analysis results of the milk somatic cell count trait of the first-litter Hu ewes, the high and low indexes of the milk somatic cell count of the adult breeding and production Hu ewes can be judged by blood sampling, DNA extraction, and KASP rapid genotyping technology of the present patent. In order to breed Hu ewe population with resistance to mastitis and low milk somatic cell count, the offspring with AA genotype at Chr1: g. 61541386 site should be continuously selected and retained in the flock, and the offspring with GG genotype at Chr1: g. 61541386 site should be continuously eliminated from the flock.

Claims

1. A method for rapidly detecting single nucleotide polymorphisms at the Chr1:g.61541386 locus in sheep using KASP for marker-assisted selection breeding of sheep, wherein the method is for marker-assisted selection breeding of low somatic cell count in sheep milk; the method comprises the following steps: Using sheep genomic DNA as a template, a partial fragment containing the A>G variant site of Chr1: g. 61541386 was amplified using specific KASP primers. The genotype of the A>G variant site of Chr1: g. 61541386 was then identified. The sheep reference genome version for Chr1: g.61541386 was ARS-UI_Ramb_v3.

0. The specific KASP primer sequences were: Site A typing primer FAM: 5'-gaaggtgaccaagttcatgctGATGGAGGAGCCTGGTGGA-3': Site G typing primer VIC: 5'-gaaggtcggagtcaacggattGATGGAGGAGCCTGGTGGG-3'; Universal primers: 5'-CCATTTCCTTCTCCAATGCATGAA-3'; The AA genotype is used as the genotype for reducing the somatic cell count trait in sheep milk; the sheep are Hu sheep.

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