SNP (Single Nucleotide Polymorphism) for selecting piglets with high diarrhea resistance, detection method and application of SNP

A technology for piglets and piglet diarrhea, which is applied in the field of SNP selection of piglets with high anti-diarrhea ability, and can solve problems such as increasing the difficulty of vaccine development, drug residues, and enhanced bacterial resistance

Pending Publication Date: 2022-04-12
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] Piglet diarrhea has caused huge economic losses to pig production. Although drug treatment and vaccination have played an important role in prevention and control, the cross-infection of diarrhea virus, the variation of pathogenic bacteria and the change of environment have greatly increased the difficulty of vaccine development, and The extensive use of antibiotics has also led to the enhancement of bacterial resistance, and also caused problems such as drug residues and food safety.

Method used

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  • SNP (Single Nucleotide Polymorphism) for selecting piglets with high diarrhea resistance, detection method and application of SNP
  • SNP (Single Nucleotide Polymorphism) for selecting piglets with high diarrhea resistance, detection method and application of SNP
  • SNP (Single Nucleotide Polymorphism) for selecting piglets with high diarrhea resistance, detection method and application of SNP

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Analysis of the promoter region of porcine STAT3 gene by PCR

[0050] 1. Genome extraction: The pig ear tissue was taken, and genomic DNA was extracted by conventional phenol-chloroform method.

[0051] 2. Primer design: Query pig STAT3 gene information (transcript ID: ENSSSCT00000018944.4) through NCBI's Ensemble database, download the 2500bp nucleotide sequence upstream of the first exon for primer design, the primers are STAT3F0 and STAT3R0, and the primer sequences are as follows :

[0052]

[0053] 3. PCR amplification: In this example, conventional PCR was used to amplify the -1134-+772 fragment of the promoter region of the STAT3 gene of Min pig. Amplification system: 100ng of genomic DNA, 25μL of 2×Taq Master Mix (Dye Plus), 10pmol of upstream and downstream primers, ddH 2 O make up to 50 μL. The amplification program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 m...

Embodiment 2

[0071] Msp I PCR-RFLP typing technique to detect the genotype of different breeds of pigs

[0072] 1. Establishment of genotyping technology:

[0073] An A / G mutation was found at -870bp before the first exon of the STAT3 gene sequence. After the second base before the SNP site was mutated from G to C, Msp I (CCGG) forced enzyme could be generated cut point. After the PCR product was digested by Msp I enzyme, three band types appeared, respectively for the three genotypes: one band that was not recognized by Msp I enzyme was recorded as AA genotype (158bp), which could be recognized by Msp I enzyme. The identified two bands were GG genotype (137bp, 21bp), and the enzyme cut into three bands was recorded as AG genotype (158bp, 137bp, 21bp).

[0074] 2. Primer Design

[0075] According to the above analysis, STAT3 genotyping primers containing the SNP (NC_010454.4:rs327720240) site were designed. The primer sequences are as follows:

[0076]

[0077] 3. PCR amplification ...

Embodiment 3

[0086] Application of the molecular marker of the present invention in the correlation analysis of diarrhea in Min pig and Landrace pig suckling piglets

[0087] The experimental population used in this example came from Landrace pigs and Min pigs born in the same period in the autumn of 2013 in Lanxi County, Heilongjiang Province. From birth to 35-day-old weaning, check the anus of each suckling piglet at 6:00 am and 2:00 pm every day to observe whether there is fecal contamination, whether the anus is red and swollen, and the defecation of the piglets, and record and score. . The stool scoring standard is: the appearance of stool is strip or granular, the degree of diarrhea is normal, and the score is 0; the stool is soft and can be formed, and the degree of diarrhea is mild, and the score is 1; There was no separation of feces and water, the degree of diarrhea was moderate, and the score was 2; the feces were liquid, shapeless, and the feces and water were separated, and t...

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Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) for selecting piglets with high diarrhea resistance, a detection method and application of the SNP, and relates to the field of animal breeding. Gene markers related to diarrhea resistance or susceptibility of suckling piglets of landrace and Min pigs are screened out, STAT3 is selected as a piglet diarrhea resistance breeding candidate gene, and the piglet diarrhea resistance or susceptibility of the suckling piglets of the landrace and the Min pigs is obtained by searching genetic variation of a pig STAT3 gene promoter region. The molecular marker and the detection method thereof are provided and applied to pig breeding, a pig molecular marker assisted breeding method is established, and a gene marker and a technical method are provided for breeding of a new pig anti-diarrhea strain. The SNP site is located in rs327720240 of a pig STAT3 gene promoter region, and the polymorphism of the SNP site is A/G. The molecular marker is applied to identification of diarrhea resistance or susceptibility of suckling piglets. The nucleotide sequence of the molecular marker is as shown in SEQ ID NO: 1. The method is applied to the breeding field.

Description

technical field [0001] The invention relates to the field of animal breeding, in particular to a SNP, a detection method and an application for selecting piglets with high anti-diarrhea ability. Background technique [0002] Piglet diarrhea has caused huge economic losses to pig production. Although the use of drug treatment and vaccination has played an important role in prevention and control, the cross-infection of diarrhea viruses, the mutation of pathogenic bacteria and changes in the environment have greatly increased the difficulty of vaccine research and development, and Extensive use of antibiotics also leads to increased drug resistance of bacteria, and also causes problems such as drug residues and food safety. Therefore, in the long run, genetically improving the resistance of pigs to pathogens can solve the problem of piglet diarrhea from the source. [0003] Signal transducer and activator of transcription 3 (STAT3) was originally discovered in mouse liver as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11
Inventor 牛步月杨秀芹陈志华王希彪狄生伟
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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