ASAP1 gene molecular marker and application thereof in sheep breeding

Abstract

The invention discloses an ASAP1 gene molecular marker and application thereof in sheep breeding, and belongs to the field of molecular biology, an SNP exists on an ASAP1 gene and is remarkably related to the sheep mycoplasma pneumonia resistance character, the SNP is located at the 23560038 site of the ninth chromosome of sheep and has A / C polymorphism, when the genotype of the SNP site is AC, the SNP site has better mycoplasma pneumonia resistance compared with AA, and when the genotype of the SNP site is not AC, the SNP site has the advantages that the SNP site has the advantages of being capable of being used for sheep breeding; the molecular marker can be used as a sheep anti-mycoplasmal pneumonia character molecular marker.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a sheep ASAP1 gene SNP molecular marker and application thereof in sheep breeding. Background Art

[0002] Sheep is one of the important sources of animal protein for humans. In the sheep farming industry, mycoplasma pneumonia is a common chronic disease in sheep herds. The growth rate of sick sheep is affected, and serious cases may cause death of sheep, which is very harmful to sheep farming. Therefore, improving the disease resistance of sheep is of great significance for sheep farming. Breeding sheep strains resistant to sheep mycoplasma pneumonia can improve the disease resistance of sheep herds. In sheep breeding, molecular markers with disease resistance can be used to identify individuals with better disease resistance for breeding, but there are currently few molecular markers suitable for selecting disease resistance traits in domestic local sheep breeds.

[0003] ASAP1 is a GTPase activating protein of ADP-ribosylation factors (Arf), which can induce the hydrolysis of GTP bound to Arf and has multiple domains such as SH3, BAR, PH, GAP and ANK (Liu et al, 2002; Luo et al, 2019). The function of ASAP1 is mainly concentrated in the regulation of endocytosis, intracellular vesicle transport and cytoskeleton movement, and is also related to tumor metastasis and spread (Tanna et al, 2019; Sabe et al, 2009). Zhang et al. found that ASAP1 can regulate intracellular inflammatory response, inhibit the activation of ASAP1 and reduce airway inflammatory response (Zhang et al, 2023). Therefore, the ability of ASAP1 to inhibit animal inflammatory response makes it a candidate gene for resisting sheep inflammation-related diseases. However, the direct effect of this gene on sheep and its mechanism of action have not been reported. Therefore, locating, discovering and verifying the molecular markers related to disease resistance of this gene can be used for molecular breeding of sheep to improve disease resistance traits. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a major effect gene locus and a corresponding detection method that can be used to screen the resistance to mycoplasma pneumonia in local sheep in China.

[0005] The technical solution of the present invention is: a method for breeding sheep with high resistance to mycoplasma pneumonia, detecting the genotype of position 23560038 of chromosome 9 of sheep, the reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1, and individuals with genotype AC at this site have higher resistance to mycoplasma pneumonia than individuals with genotype AA.

[0006] The invention discloses an application of a material for detecting the genotype of a SNP site in auxiliary breeding of sheep anti-mycoplasma pneumonia traits, wherein the SNP site is located at position 23560038 of chromosome 9 of sheep, located at position 273 of the nucleotide sequence described in SEQ ID No. 1, and has an A / C polymorphism.

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

[0008] A primer pair, the nucleotide sequence of the primer pair is shown as SEQ ID No.2 and SEQ ID No.3.

[0009] A kit comprising the primer pair described above.

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

[0011] The present invention finds that there is a SNP on the ASAP1 gene that is significantly correlated with the sheep's anti-mycoplasma pneumonia trait, and can be used as a molecular marker for the sheep's anti-mycoplasma pneumonia trait. The sheep individuals identified by this method have relatively strong anti-mycoplasma pneumonia ability, and can be used to breed new sheep strains with anti-mycoplasma pneumonia performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Significant signal loci associated with anti-mycoplasma pneumonia obtained through gene association analysis;

[0013] Figure 2 PCR result graph including the target SNP site;

[0014] Figure 3 The sequencing results are a comparison of the CT values ​​of Mycoplasma pneumonia between Hu sheep containing this molecular marker and wild-type individuals. DETAILED DESCRIPTION

[0015] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0016] Example 1 Discovery of SNP Molecular Markers of ASAP1 Gene

[0017] A total of 538 Hu sheep tested for mycoplasma pneumonia were resequenced, and combined with the load data of Mycoplasma pneumoniae in sheep nasal swabs obtained by detection, the ASAP1 gene ( Figure 1). There is a SNP on the ASAP1 gene that is significantly associated with the sheep's anti-mycoplasma pneumonia trait. The SNP is located at position 23560038 on chromosome 9 of sheep (the reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1) and has an A / C polymorphism. When the genotype of the SNP site is AC, it has better anti-mycoplasma pneumonia performance than AA, and can be used as a molecular marker for sheep's anti-mycoplasma pneumonia trait.

[0018] Example 2 Detection of SNP Molecular Markers

[0019] (1) The SNP is located at position 273 of the nucleotide sequence shown in SEQ ID No. 1. Based on the upstream and downstream sequences of the SNP site, specific primers for amplifying the fragment were designed:

[0020] Upstream primer ASAP1F1: 5′-TCCGTGAGTTAATGGGCCTGTC-3′ (SEQ ID No. 2)

[0021] Downstream primer ASAP1R1: 5′-GCCATACCAATCCATGAAGCCAG-3′ (SEQ ID No. 3)

[0022] (2) PCR amplification was performed on the sheep DNA sample using the specific primers of (1) to obtain an amplification product of the region containing the mutation. The amplification product was 468 bp ( Figure 2 ).

[0023] The amplification system is as follows:

[0024] Table 1 ASAP1 amplification system

[0025]

[0026] 2× Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0027] The amplification conditions were as follows: amplification was performed using a PCR amplification instrument, the first stage: pre-denaturation at 95°C for 5 min; the second stage: denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 34 cycles; the third stage: final extension at 72°C for 5 min, and storage at 4°C.

[0028] (3) The amplified product is subjected to Sanger sequencing to obtain the genotype of the SNP site.

[0029] Example 3 Verification of SNP Molecular Markers

[0030] The genomes of 119 sheep (Hu sheep breed) were amplified, and the amplification primers, conditions, and systems were the same as in Example 2. The results of PCR product sequencing are shown in Table 2. Seven sheep were heterozygous individuals (genotype AC) containing the SNP site, and 112 were wild-type individuals (genotype AA, consistent with the reference genome), and no homozygous mutant individuals with a genotype of CC were found.

[0031] Table 2 Typing results of different genotypes

[0032]

[0033] The CT values ​​of all individual Mycoplasma pneumoniae tests (CT values ​​exceeding 32 were negative) were compared, where the CT value size reflected the copy number of the Mycoplasma pneumoniae genome in sheep.

[0034] The comparison results are as follows Figure 3 As shown, the CT value of heterozygous individuals containing this molecular marker is higher than that of wild-type individuals, that is, the copy number of the Mycoplasma pneumonia genome is lower, which is significantly different, indicating that the Mycoplasma ovis pneumonia load of individuals with genotype AC is low, and this type of individuals has a stronger ability to resist Mycoplasma pneumonia.

Claims

1. A method for breeding sheep with high resistance to mycoplasma pneumonia, characterized in that: The genotype of position 23560038 on chromosome 9 of sheep was tested. The reference genome version was ARS-UI_Ramb_v2.0, GCF_016772045.

1. Individuals with genotype AC at this site had higher anti-mycoplasma pneumonia performance than those with genotype AA.

2. Application of a material for detecting the genotype of a SNP site in assisting breeding of sheep for anti-mycoplasma pneumonia traits, wherein the SNP site is located at position 23560038 of chromosome 9 of sheep, located at position 273 of the nucleotide sequence described in SEQ ID No. 1, and has an A / C polymorphism.

3. The use according to claim 2, characterized in that: The substance is a primer pair, and the nucleotide sequence of the primer pair is shown as SEQ ID No.2 and SEQ ID No.

3.

4. A primer pair, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID No.2 and SEQ ID No.

3.

5. A kit, characterized in that: Containing the primer pair according to claim 4.

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