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Identification and application of SNP in the fourth exon region of porcine incenp

A technique for boars and large white pigs, applied in DNA/RNA fragmentation, recombinant DNA technology, determination/inspection of microorganisms, etc.

Active Publication Date: 2021-08-13
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the SNP in the boar INCENP exon region

Method used

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  • Identification and application of SNP in the fourth exon region of porcine incenp
  • Identification and application of SNP in the fourth exon region of porcine incenp
  • Identification and application of SNP in the fourth exon region of porcine incenp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Extraction of Boar Semen Genomic DNA Using Phenol Extraction

[0022] Inject the fresh semen of the boar collected on the spot (from the Yaji Mountain Pig Artificial Insemination Center of Guangxi Yangxiang Pig Gene Technology Co., Ltd., the American Duroc, Landrace and Large White breeds, the same below) into 10ml centrifuge tubes and stored frozen at -20°C. Transported to the laboratory below 0°C, then thawed in a water bath, and extracted boar semen genomic DNA. Specific steps are as follows:

[0023] (1) Take 1ml of pig semen, put it in a 2ml centrifuge tube, centrifuge at 5000rpm for 7min, discard the supernatant.

[0024] (2) Add 1000ul sperm washing solution (1M Tris-Hcl (PH=8.0) 10ml, 0.5M EDTA (PH=8.0) 20ml, 5M NaCl 100ml to each centrifuge tube, add ddH 2 (870ml, mix well, conventional high-pressure steam sterilization, store at room temperature) or normal saline, blow and beat repeatedly, centrifuge at 12000rpm for 7min, and discard the supernata...

Embodiment 2

[0033] Embodiment 2: the acquisition of boar INCENP fourth exon region-specific DNA fragment and the establishment of single nucleotide polymorphism (SNP) detection method

[0034] Select pigs of foreign blood, such as American "Duroc", "Landrace", and "Large White" (from the Yaji Mountain Pig Artificial Insemination Center, a subsidiary of Guangxi Yangxiang Pig Gene Technology Co., Ltd., which are routinely reported American breeds , the implementation of the present invention is not limited to the above-mentioned varieties) take the mRNA sequence of pig INCENP gene (GeneBank accession number is NC_010444) as seed, compare in the genome of pig, design following primers according to its fourth exon sequence:

[0035] Forward primer F: 5'CTCCGCAGCAAGGACAAGG 3',

[0036] Reverse primer R: 5'AGGCACAAAGGACCACCCAC 3';

[0037] Taking Duroc pig, Landrace pig, and Large White pig blood genome DNA as templates respectively, carry out PCR amplification with the above-mentioned primer ...

Embodiment 3

[0047] Example 3: Association analysis and application of molecular markers cloned in the present invention and reproductive traits of boars

[0048] Statistics of Genotype Frequency and Allele Frequency

[0049] Genotype frequency: refers to the proportion of a specific genotype in a population to all genotypes in the population. The statistical method of genotype frequency is: genotype frequency = number of individuals of this genotype / total number of measured populations

[0050] Allele frequency: refers to the relative proportion of a gene in a population to the total genes at that locus. Statistical method of allele frequency: genotype frequency of homozygote of this gene + genotype frequency of heterozygote containing this gene / 2

[0051] Table 3 The genotype frequency and allele frequency of the SNP in the fourth exon region of INCENP in two breeds of pigs

[0052]

[0053] In order to determine whether the SNP in the fourth exon region of the pig INCENP gene is r...

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Abstract

The invention belongs to the field of porcine molecular marker preparation, and in particular relates to the identification and application of SNP in the fourth exon region of porcine INCENP. The present invention obtains a SNP molecular marker associated with boar reproductive traits, its nucleotide sequence is shown in SEQ ID NO: 1, or SEQ ID NO: 2 or SEQ ID NO: 3, in the SEQ ID NO : 1, SEQ ID NO: 2, SEQ ID NO: 3 there is a mutation of C / T, C / T and T / C respectively at 327bp of the sequence shown in SEQ ID NO: 2, and the mutation of the above allele causes the polymorphism of Tsp45I-RFLP sex. The molecular markers were used to analyze the association with reproductive traits of boars in Landrace and Large White pigs. The typing detection method of the molecular marker is also disclosed, which provides a new marker for the reproductive traits of boars and can be used for the evaluation of reproductive traits of boars and genetic improvement.

Description

technical field [0001] The invention relates to the fields of pig breeding and molecular marker-assisted selection of pigs, in particular to the identification and application of SNP in the exon region of pig INCENP as a molecular marker for reproductive traits of boars. Background technique [0002] Molecular marker-assisted selection (MAS, marker-assisted selection) is a new technology produced with the rapid development of modern molecular biology technology, which can quickly and accurately analyze the genetic composition of individuals at the molecular level, so as to realize the genotype Direct selection, molecular breeding. Molecular markers are directly expressed in the form of DNA, which can be detected in all tissues and developmental stages of organisms, and are not restricted by seasonal environments; polymorphisms are abundant and large in number; most of them are co-dominant, and can distinguish homozygous and heterozygous fit. Therefore, molecular marker-ass...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/156
Inventor 徐德全张龙刘敏夏文周昌繁
Owner HUAZHONG AGRI UNIV
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