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Method of screening SNP (single nucleotide polymorphism) sites in HSTN (histatherin) gene of cow with mastitis resistance and kit

A mastitis and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., to reduce detection costs and improve detection efficiency

Active Publication Date: 2012-07-25
DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The HSTN gene is an important gene that affects bovine mastitis traits, and its single nucleotide polymorphism (SNP) is the genetic basis that leads to differences in anti-mastitis traits among different cattle individuals, but using its SNP and haplotype combination and The relationship between dairy cows' resistance to mastitis No report on the kit for detecting the resistance traits of dairy cows

Method used

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  • Method of screening SNP (single nucleotide polymorphism) sites in HSTN (histatherin) gene of cow with mastitis resistance and kit
  • Method of screening SNP (single nucleotide polymorphism) sites in HSTN (histatherin) gene of cow with mastitis resistance and kit
  • Method of screening SNP (single nucleotide polymorphism) sites in HSTN (histatherin) gene of cow with mastitis resistance and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Haplotype combination and determination of mutation sites

[0044] The present invention adopts PCR sequencing and PCR-RFLP technology to screen the mutation of dairy cattle HSTN gene, and determines three mutation sites of 635, 1607 and 12624 through enzyme-cut electrophoresis bands and comparison of sequencing results. Haplotypes were constructed using Shesis software.

[0045] 1.1 Collection of milk cow jugular vein blood (the Holstein cow jugular vein blood used in the present invention is taken from large-scale cattle farms in Shandong Province, including Jinan Jiabao, Qingdao Yimu, etc., which are the direct source and original source of the blood sample used in the present invention), ACD anticoagulation, using the conventional phenol / chloroform extraction method to extract genomic DNA, DNA samples were diluted to 50ng / μL for later use. A total of 500 experimental cows came from large-scale dairy farms in Shandong Province.

[0046] 1.2 Primer design ...

Embodiment 2

[0062] Example 2 Correlation between HSTN Gene Haplotype Combination and Dairy Cow Mastitis Resistance

[0063] Determination of somatic cell score: Collect 40 mL of milk sample, add 0.03 g of potassium dichromate, mix gently, and then send it to the DHI laboratory of the Dairy Center of Shandong Academy of Agricultural Sciences to use the Foss 6000 milk component and somatic cell online analysis system (Foss MilkScan FT 6000, Fossmatic 5000) to measure the number of somatic cells. According to the formula SCS=log2(SCC / 100)+3 (wherein SCS-somatic cell score; SCC-somatic cell number, unit thousand / mL), the individual's somatic cell score was calculated.

[0064]The least square analysis of variance adopts the ANOVA-Linear Models program of SAS 9.0 (Statistical Analysis System) software to establish the following model, and perform the least square analysis and significance test on the somatic score and haplotype. The model is:

[0065] Y ijkl =μ+G i +E j +S k +P l +e ijk...

Embodiment 3

[0072] Example 3 Detection Kit

[0073] A kit for screening dairy cows based on HSTN gene haplotype combination markers, as described in Example 1, the haplotypes constructed by the 635 A→T mutation, 1607 T→C and 12624 T→G mutations in NC_007304.5 Combinations were strongly associated with mastitis resistance traits in dairy cows. Therefore, HSTN gene-specific primers can be designed based on these three SNP sites for amplification and detection.

[0074] Prepare the kit (100 times) for detecting mastitis resistance traits of dairy cows, the composition is as shown in Table 4:

[0075] Table 4

[0076]

[0077] The bovine blood was collected, the total DNA was extracted, and the reaction was carried out according to the method described in Example 1. Dilute the PCR primers in the kit to 10 μmol / L, and use the extracted DNA as a template to carry out PCR reaction with the above kit. PCR reaction conditions: Denaturation at 95°C for 5min, denaturation at 94°C for 30s, ann...

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Abstract

The invention discloses a method of screening and detecting SNP (single nucleotide polymorphism) sites of a cow with mastitis resistance, wherein the SNP sites are basic groups at the 635th site, the 1607th site and the 12624th site of an HSTN (histatherin) gene of the cow. The method comprises the steps of determining an HSTN gene haplotype combination by extraction of genomic DNA of the cow, and forecasting the mastitis resistance of the cow. When the HSTN gene haplotype combination is H2H2 (AATTGG), the mastitis resistance of the cow is the strongest; and when the HSTN gene haplotype combination is H3H7 (TTCCTG), the mastitis resistance of the cow is the weakest. The method provided by the invention has the advantages that the correlation between three polymorphism site combinations in the HSTN gene and the mastitis resistance of the cow is clarified for the first time, a method of forecasting the mastitis resistance of the cow is provided, a kit used for the method is also provided, and a new molecular labeling method is provided for marker-assisted selection and selected mating of the cow.

Description

technical field [0001] The invention belongs to the technical field of livestock molecular biology, and relates to the screening of HSTN gene SNP sites of mastitis-resistant cows, an application method and a kit. Background technique [0002] Cow mastitis (Mastitis) refers to an inflammatory change that occurs when the mammary glands of dairy cows are subjected to physical and chemical stimulation, especially the invasion of microorganisms. It not only affects the quality of milk, but also reduces milk production, causing serious damage to the development of the dairy industry. harm. According to reports, the positive rate of mastitis in my country's cattle herds can be as high as 40%-80%. Dairy cow mastitis has become the number one problem plaguing my country's dairy farming industry. Therefore, controlling its incidence and reducing the cost of treating mastitis are important Improving the economics of dairy producers is very important. [0003] Cow mastitis is divided i...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 鞠志花黄金明王长法李秋玲齐超张燕李建斌侯明海仲跻峰
Owner DAIRY CATTLE RES CENT SHANDONG ACADEMY OF AGRI SCI
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