Znf10 gene molecular marker and application thereof in sheep breeding

Abstract

The application discloses a ZNF10 gene molecular marker and application thereof in sheep breeding, and belongs to the field of molecular biology. A SNP existing on a ZNF10 gene is significantly related to a mycoplasmal pneumonia resistance trait of sheep. The SNP is located at a position of 44270644 of a 17th chromosome of the sheep, has A / G polymorphism, and when a genotype of the SNP site is AG, the sheep has better mycoplasmal pneumonia resistance performance than AA, and can be used as a molecular marker of the mycoplasmal pneumonia resistance trait of the sheep.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a sheep ZNF10 gene SNP molecular marker and its application in sheep breeding. Background Technology

[0002] Mycoplasma pneumoniae in sheep is a chronic disease that affects sheep growth and can lead to death in severe cases. Disease resistance is a crucial indicator in sheep breeding, and developing sheep strains resistant to mycoplasma pneumoniae can improve the overall disease resistance of sheep populations, thus increasing production efficiency and industrialization. If disease resistance is to be applied to sheep breeding, molecular markers are needed to identify individuals with good disease resistance for breeding purposes. Currently, there are relatively few molecular markers suitable for selecting disease resistance traits for local Chinese sheep breeds.

[0003] ZNF10 (Zinc finger protein 10) is a member of the KRAB-type zinc finger protein family. The KRAB domain, located at the N-terminus of the protein, acts as a transcriptional repressor by binding to co-repressor proteins; while the C-terminal C2H2 zinc finger domain binds to DNA (Urrutia et al., 2003; Ecco et al., 2017). Studies have reported that SARS-CoV-2 infection increases the expression of the ZNF protein family, enhances interferon signaling, or directly promotes the activation and maturation of various immune cells to combat SARS-CoV-2 infection (Qin et al., 2021). In small ruminant measles virus (SRMV) infection, zinc finger proteins confer resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication (Tang et al., 2021). Nishitsuji et al. discovered that the KRAB domain of ZNF10 can bind to TRIM28, SETDB1, and HP1-γ to inhibit HIV-1LTR-mediated transcription, thus exerting an anti-HIV effect (Nishitsuji et al., 2015). Therefore, the ZNF10 gene may be related to disease resistance in animals, but its direct effects on sheep and its mechanism of action have not been reported. Discovering and validating molecular markers in the ZNF10 gene associated with sheep disease resistance could make this site a target for selective breeding to improve disease resistance traits in sheep. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a major gene locus and corresponding detection method for screening resistance to Mycoplasma pneumoniae in local Chinese sheep.

[0005] The technical solution of the present invention is as follows: a method for breeding sheep with high resistance to mycoplasma pneumonia, wherein the genotype at position 44270644 on chromosome 17 of sheep is detected, with the reference genome version being ARS-UI_Ramb_v2.0, GCF_016772045.1. Individuals with the genotype AG at this locus have higher resistance to mycoplasma pneumonia than individuals with the genotype AA.

[0006] The application of substances for detecting SNP locus genotypes in sheep growth performance-assisted breeding, wherein the SNP locus is located at position 44270644 on sheep chromosome 17, at position 495 of the nucleotide sequence described in SEQ ID No. 1, and exhibits G / A polymorphism.

[0007] Furthermore, the substance is a primer pair, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0008] Primer pair, the nucleotide sequences of which are shown in SEQ ID No. 2 and SEQ ID No. 3.

[0009] A reagent kit containing the primer pairs described above.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] This invention discovered a SNP in the ZNF10 gene that is significantly associated with the trait of resistance to mycoplasma pneumoniae in sheep, and can serve as a molecular marker for this trait. Sheep individuals identified using this method possess relatively strong resistance to mycoplasma pneumoniae and can be used to breed new sheep strains with resistance to mycoplasma pneumoniae. Attached Figure Description

[0012] Figure 1 Significant signaling sites associated with anti-mycoplasma pneumonia were obtained through gene association analysis;

[0013] Figure 2 A graph showing PCR results including the target SNP site;

[0014] Figure 3 Sanger sequencing results of PCR product fragments;

[0015] Figure 4 The sequencing results show a comparison of Mycoplasma pneumoniae CT values ​​between Hu sheep containing this molecular marker and wild-type individuals. Detailed Implementation

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0017] Example 1: Discovery of ZNF10 gene SNP molecular markers

[0018] Resequencing was performed on 538 Hu sheep individuals tested positive for mycoplasma pneumonia. Combined with data on the viral load of Mycoplasma pneumoniae in sheep nasal swabs, genome-wide association analysis was used to locate the ASAP1 gene. Figure 1 A SNP on the ASAP1 gene is significantly associated with resistance to mycoplasma pneumoniae in sheep. This SNP is located at position 44270644 on sheep chromosome 17 (reference genome version: ARS-UI_Ramb_v2.0, GCF_016772045.1) and exhibits A / G polymorphism. When the genotype at this SNP site is AG, it has better resistance to mycoplasma pneumoniae than AA and can be used as a molecular marker for mycoplasma pneumoniae resistance in sheep.

[0019] Example 2: Detection of SNP molecular markers

[0020] (1) The SNP is located at position 495 of the nucleotide sequence shown in SEQ ID No. 1. Based on the upstream and downstream sequences of this SNP site, specific primers for amplifying this fragment were designed:

[0021] Upstream primer ZNF10F1: 5′-CAGTGGCCATCAGGAAAGCTG-3′ (SEQ ID No. 2)

[0022] Downstream primer ZNF10R1: 5′-CCACAGTCATGACATTCGTATGGC-3′ (SEQ ID No. 3)

[0023] (2) Using the specific primers from (1), PCR amplification was performed on the sheep DNA sample to obtain an amplified product containing the mutated region. The amplified product was 795 bp. Figure 2 ).

[0024] The amplification system is as follows:

[0025] Table 1 ASAP1 amplification system

[0026]

[0027] 2× Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novizan Biotechnology Co., Ltd., catalog number P525-02.

[0028] The amplification conditions were as follows: Amplification was performed using a PCR amplification instrument. The first stage was pre-denaturation at 95℃ for 5 min; the second stage was denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 500 s, for 34 cycles; the third stage was final extension at 72℃ for 5 min, and storage at 4℃.

[0029] (3) The amplified products were subjected to Sanger sequencing. Figure 3 ), to obtain the genotype of the SNP locus.

[0030] Example 3: Validation of SNP molecular markers

[0031] The genomes of 120 sheep (Hu sheep breed) were amplified using the same primers, conditions, and system as in Example 2. After sequencing the PCR products, 6 sheep were found to be heterozygous individuals containing the SNP locus (genotype AG), 114 were wild-type individuals (genotype AA, consistent with the reference genome), and no homozygous mutant individuals with genotype GG were found.

[0032] The CT values ​​for Mycoplasma pneumoniae detection were compared among all individuals (where a CT value greater than 32 was considered negative), where the CT value reflects the copy number of the Mycoplasma pneumoniae genome in sheep.

[0033] The comparison results are as follows Figure 4 As shown, heterozygous individuals containing this molecular marker had higher CT values ​​than wild-type individuals, indicating a lower copy number of the Mycoplasma pneumonia genome, which was significantly different. This suggests that individuals with the AG genotype had a lower Mycoplasma pneumoniae load in sheep and thus had stronger resistance to Mycoplasma pneumoniae.

Claims

1. Application of substances for detecting SNP locus genotypes in the preparation of reagents for breeding sheep with high resistance to mycoplasma pneumoniae; the SNP locus is located at position 44270644 on sheep chromosome 17, reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1, located at position 495 of the nucleotide sequence shown in SEQ ID No.1, and has G / A polymorphism. Individuals with the genotype AG at this locus have higher resistance to mycoplasma pneumoniae than individuals with the genotype AA.

2. The application according to claim 1, characterized in that, The substance is a primer pair, and the nucleotide sequences of the primer pair are shown in SEQ ID No. 2 and SEQ ID No. 3.

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