Genetic mark using SNP of third exon of CATSPER4 gene as sperm quality property of pigs

A technology of genetic markers and quality traits, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of low heritability of reproductive traits in boars

Inactive Publication Date: 2019-06-04
HUAZHONG AGRI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The low heritability of reproductive traits in boars makes it difficult fo...

Method used

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  • Genetic mark using SNP of third exon of CATSPER4 gene as sperm quality property of pigs
  • Genetic mark using SNP of third exon of CATSPER4 gene as sperm quality property of pigs
  • Genetic mark using SNP of third exon of CATSPER4 gene as sperm quality property of pigs

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1: Obtaining of porcine CATSPER4 gene fragment and establishment of polymorphism detection method

[0020] Log in to the Ensembl database to download the porcine CATSPER4 gene sequence (the accession number is ENSSSCG00000026526), ​​find the screened SNP site in the sequence, and use Primer Premier 5 software to design primers. The sequence of the forward primer is shown in SEQ ID NO: 2 in the sequence table, that is, CATSPER4-AlwNI-F: GCA ATG ACC ACAGCG AAG A, and the sequence of the reverse primer is shown in SEQ ID NO: 3 in the sequence table, that is, CATSPER4-AlwNI -R: TGC TCAGGC CCG TTA GAT C. Amplified to obtain a 410bp target fragment (the sequence of this fragment is shown in SEQ ID NO: 1 (see figure 2 ). The PCR reaction system is 25 μl, including 1 μl template DNA, 0.5 μl each primer, 12.5 μl PCR Mix, and finally add 10.5 μl deionized water to a total volume of 25 μl; PCR reaction program: 94 ° C pre-denaturation for 5 minutes; then Denaturation a...

Embodiment 2

[0022] Example 2: Polymorphic distribution of genetic markers prepared by the present invention in different pig herds

[0023] The extraction of pig genomic DNA (sample is shown in Table 1) was carried out with reference to the method reported by Xiong Yuan, "Introduction to Pig Biochemistry and Molecular Genetic Experiments", China Agricultural Press, 1999.

[0024] The PCR-AlwNI-RFLP polymorphism of porcine CATSPER4 gene was detected in three foreign blood-related pig populations, and the detection results are shown in Table 1.

[0025] Table 1 Distribution results of porcine CATSPER4 gene PCR-AlwNI-RFLP in different pig breeds

[0026]

[0027] Note: The above group materials are all species publicly promoted in China.

[0028] The results showed that there were 3 genotypes in Duroc, Large White and Landrace pigs. In the Duroc pig population, the G allele frequency is 0.83, which is the dominant allele; in the Large White pig population, the G allele frequency and T all...

Embodiment 3

[0029] Example 3: Association analysis of genetic markers prepared by the present invention and semen quality traits

[0030] In order to determine whether the pig CATSPER4 gene PCR-AlwNI-RFLP is related to the difference in the quality of pig semen, the Duroc pigs, Large White pigs and Landrace pigs bred in the breeding farm of Guangxi Yangxiang Co., Ltd. were selected as test materials, and the pigs established in Example 1 were used. AlwNI-RFLP method was used to detect polymorphisms, and the correlation between different genotypes of pig AlwNI-RFLP and pig semen quality traits was analyzed. The SAS statistical software (SAS Institute Inc, Version 9.4) GLM program was used for single-marker analysis of variance, and the REG program was used to calculate the additive effect and dominant effect of genes, and the significance test was carried out. The model used was:

[0031] Y ij =μ+G i +F j +e ij

[0032] Y ij is the trait phenotype value, μ is the average value, G i ...

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Abstract

The invention belongs to the technical fields of preparation and application of a genetic mark of pigs, and relates to a genetic mark using SNP of a third exon of a CATSPER4 gene as sperm quality property of pigs. The genetic mark is cloned from a CATSPER4 gene exon 3 of the pigs, and the nucleotide sequence is shown as SEQID NO:3. The invention discloses an SNP site on the CATSPER4 gene exon 3 ofthe pigs and a detection method of an SNP subtype. Allelic gene mutation exists on the CATSPER4 gene exon 3, and the mutation causes AlwNI-RFLP polymorphism. The screening step comprises: extractinggenome DNA from pig blood, seeking the SNP site from sequencing results, determining the incision enzyme of the SNP site, and through primer design, PCR amplification and single nucleotide polymorphism detection, enabling the genotype and the sperm quality property to be subjected to correlation analysis. The screened mark can be used for assisting in selection of the mark of the pigs.

Description

technical field [0001] The invention relates to the technical field of preparation of pig genetic markers, in particular to a SNP of the third exon of CATSPER4 gene as a genetic marker for pig semen quality traits and its application, which includes a detection method and application of pig CATSPER4 gene mutation sites. Background technique [0002] The reproductive performance of boars plays an important role in the size of the herd and the efficiency of pig farms. Generally, the reproductive performance of boars is measured by identifying the quality of semen. The indicators of semen quality include sperm concentration, sperm motility, abnormal sperm rate and sperm morphology, kinetic parameters and DNA integrity. Semen quality is mainly determined by two factors: genetic effect and environmental effect. Environmental factors can be artificially controlled and improved, but only continuous improvement of genetic effect is the key to improving semen quality and boar reprodu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/683C12Q1/686C12N15/11
Inventor 李凤娥赵丽萍宋慧彬冯越陈大可
Owner HUAZHONG AGRI UNIV
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