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SNP molecular marker related to reproductive characters of Chinese Holstein cow and applications of SNP molecular marker

A molecular marker and molecular marker-assisted technology, applied in the field of molecular biology, can solve the problems of disturbing the body's energy homeostasis, insulin sensitivity, affecting the transcriptional activity and expression of PPARγ, binding and covalent modification activation, etc., to accelerate genetic progress. , The method is accurate and reliable, and the detection cost is low.

Inactive Publication Date: 2017-10-20
GANSU NORMAL UNIV FOR NATTIES
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Problems solved by technology

If the nucleotide sequence of the PPARG gene is mutated, it may affect the binding of PPARγ to the ligand and the activation of covalent modification, reduce the affinity of PPARγ / RXRα and the upstream PPRE of the energy homeostasis-related gene promoter, and then affect the PPARγ. Transcriptional activity and expression, disturbing the body's energy homeostasis and insulin sensitivity, affecting reproductive physiological processes such as estrus, ovulation and embryonic development

Method used

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  • SNP molecular marker related to reproductive characters of Chinese Holstein cow and applications of SNP molecular marker
  • SNP molecular marker related to reproductive characters of Chinese Holstein cow and applications of SNP molecular marker
  • SNP molecular marker related to reproductive characters of Chinese Holstein cow and applications of SNP molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Determination of SNP markers related to reproductive traits of Chinese Holstein dairy cows and establishment of a method for detecting the loci

[0036] 1. Extract the genomic DNA from the blood of Chinese Holstein cows to be tested

[0037] Blood samples from 293 Chinese Holstein cows in different pastures were collected from the jugular vein, anticoagulated with ACD, and stored at -20°C for later use. The genomic DNA in bovine blood exists in the solution in the centrifuge tube, which can be stored at 4°C for later use or at -20°C for long-term storage.

[0038] 2. Amplify the nucleotide fragment containing the SNP site

[0039] Primers were designed according to the sequence of the PPARG gene (GenBank No.NC_007320.6) included in the NCBI database, including forward primer F: 5′-TGTAACTCAACCTCCTGTT-3′ and reverse primer R: 5′-AAGATGCTGTCAGTGAACT-3′, using the above genomic DNA As a template, the nucleotide fragment where the SNP to be detected is located i...

Embodiment 2

[0047] Example 2 Correlation analysis and detection application of different genotypes and reproductive traits of Chinese Holstein cows

[0048] According to the method of Example 1, 293 Chinese Holstein dairy cows were detected by PCR-SSCP, and the analysis results of the 118bp site of the sequence shown in SEQ ID NO.1 of the PPARG gene are shown in Table 1.

[0049] Table 1 Genetic polymorphism analysis of SNP loci in Chinese Holstein cow population

[0050]

[0051] It can be seen from Table 1 that GG individuals have the highest frequency in the test population, and G is the dominant allele. He was 0.4147, PIC was 0.3270, and was moderately polymorphic. χ obtained by Pearson chi-square test 2 19.71>χ0.01 2 (df=2)=9.21, then P<0.01, the test population deviates from the Hardy-Weinberg equilibrium state very significantly at this site, indicating that its mutation is affected by factors such as drift, selection and introduction.

[0052] Use SPSS software to call the ...

Embodiment 3

[0068] Example 3 Assembly and application of a kit for identifying Chinese Holstein cows with high reproductive performance

[0069] The components in the kit are as follows: 100ng / μL bovine blood DNA; upstream and downstream primers, the sequences of which are shown in SEQ ID NO.2-3 respectively, 2.5mmol / L dNTPs; 2.5U / μL Taq DNA polymerase; 10×Taq Buffer (Containing 15mmol / LMgCl 2 );Ultra-pure water.

[0070] F: 5'-TGTAACTCAACCTCCTGTT-3' (SEQ ID NO.2)

[0071] R: 5'-AAGATGCTGTCAGTGAACT-3' (SEQ ID NO.3)

[0072] The total PCR reaction system in the kit is 25μL, including 1μL of genomic DNA, 10×buffer (15mmol / LMgCl 2 ) 2.5μL, dNTP (2.5mmol / L) 2μL, upstream and downstream primers (12.5pmol / μL) each 0.5μL, Taq DNA polymerase (2.5U / μL) 0.5μL, ddH 2 O 18.0 μL. PCR reaction program: pre-denaturation at 95°C for 3min; denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 45s, 35 cycles; final extension at 72°C for 10min.

[0073] The product obtained fr...

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Abstract

The invention provides an SNP molecular marker related to reproductive characters of a Chinese Holstein cow and an application of the SNP molecular marker and belongs to the technical field of molecular breeding. A specific fragment is amplified from a PPARG gene of the cow, and a nucleotide sequence is as shown in a sequence table SEQ ID NO.1, wherein single-base mutation of G-T is formed at the 118th bp of the sequence table SEQ ID NO.1. The number of postpartum first breeding days, the number of nonpregnant days and the conception index of the Chinese Holstein cow employing G as a base on an SNP marker site are significantly lower than those of the Chinese Holstein cow employing T as the base. The invention further provides a primer for detecting the SNP marker and a kit containing the primer. The invention further provides an application of the SNP marker in identification of the dominant variety of the Chinese Holstein cow with high reproductive performance and an application in molecular marker-assisted breeding of the Chinese Holstein cow. The SNP molecular marker has the advantages of being simple in operation, high in sensitivity, high in accuracy and low in detection cost.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a SNP molecular marker related to the reproductive traits of Chinese Holstein cows, a kit for detecting the molecular marker, and applications of the SNP molecular marker and the kit. Background technique [0002] Marker Assistant Select (MAS) has a larger amount of information than conventional genetic assessment methods due to the full use of genetic markers, phenotype and pedigree information, and can improve the accuracy of individual genetic assessment. Meuwissen et al. (2001) proved by simulation that the accuracy of predicting breeding values ​​based on marker information can reach 85%. In the absence of progeny determination records, it is of great significance to estimate the breeding value with such high accuracy. The application of MAS in cattle breeding has many advantages: it is not easily affected by the environment, has no gender and age restrictions, al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 马彦男贺鹏迦马正才董艳娇朱静马永生
Owner GANSU NORMAL UNIV FOR NATTIES
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