An ASAP1 gene molecular marker and its application in sheep breeding

Abstract

The present invention discloses an ASAP1 gene molecular marker and its application in sheep breeding, belonging to the field of molecular biology. There is a SNP on the ASAP1 gene that is significantly associated with the trait of sheep resistance to mycoplasma pneumonia. This SNP is located at position 23560038 on sheep chromosome 9 and has A / C polymorphism. When the genotype of this SNP locus is AC, it has better performance in resisting mycoplasma pneumonia than AA, and can be used as a molecular marker for the trait of sheep resistance to mycoplasma pneumonia.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to an SNP molecular marker of sheep ASAP1 gene and its application in sheep breeding. Background Art

[0002] Sheep are one of the important sources of human animal protein. In the sheep breeding industry, mycoplasmal pneumonia is a common chronic disease in the sheep population. The growth rate of diseased sheep is affected, and in severe cases, it will cause the death of sheep, which has a great harm to sheep breeding. Therefore, improving the disease resistance of sheep is of great significance for sheep breeding. Breeding sheep strains resistant to mycoplasmal pneumonia can improve the disease resistance of the sheep population. In sheep breeding work, molecular markers with disease resistance can be used to identify individuals with better disease resistance for breeding. However, there are currently few molecular markers suitable for selecting disease-resistant traits of domestic local sheep breeds in China.

[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 functions of ASAP1 are mainly concentrated in the regulation of intracellular endocytosis, intracellular membrane vesicle trafficking, and cytoskeleton movement, and are 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 responses, and inhibiting the activation of ASAP1 can reduce airway inflammatory responses (Zhang et al, 2023). Therefore, the ability of ASAP1 to inhibit animal inflammatory responses makes it a candidate gene for resisting sheep inflammatory-related diseases. However, the direct effect of this gene on sheep and its mechanism of action have not been reported. Therefore, localizing, discovering, and validating molecular markers related to sheep disease resistance in this gene can be used for molecular breeding of sheep to improve disease-resistant traits. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a major gene locus and corresponding detection method for screening the disease resistance of Chinese local sheep to mycoplasmal pneumonia.

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

[0006] Application of substance for detecting genotype of SNP locus in assisted breeding of sheep against mycoplasmal pneumonia traits, wherein the SNP locus is located at position 23560038 on chromosome 9 of sheep, located at position 273 of the nucleotide sequence shown in SEQ ID No.1, and has A / C polymorphism.

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

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

[0009] A kit contains the above-mentioned primer pair.

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

[0011] The present invention discovers that there is an SNP on the ASAP1 gene that is significantly associated with the traits of sheep against mycoplasmal pneumonia and can be used as a molecular marker for the traits of sheep against mycoplasmal pneumonia. The sheep individuals identified by this method have relatively strong ability to resist mycoplasmal pneumonia and can be used to breed new sheep strains with the performance of resisting mycoplasmal pneumonia. Description of the Drawings

[0012] Figure 1 Significant signal loci related to mycoplasmal pneumonia resistance obtained through gene association analysis;

[0013] Figure 2 PCR result diagram containing the target SNP locus;

[0014] Figure 3 Sequencing results are the comparison of CT values of mycoplasmal pneumonia between Hu sheep and wild-type individuals containing this molecular marker. Detailed Embodiments

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

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

[0017] By resequencing 538 individuals of Hu sheep detected for mycoplasmal pneumonia and combining the data of the load of Mycoplasma pneumoniae in sheep nasal swabs obtained by detection, the ASAP1 gene was located by using the genome-wide association analysis method ( Figure 1)。There is a SNP on the ASAP1 gene that is significantly associated with the trait of sheep's resistance to Mycoplasma pneumonia. This SNP is located at position 23560038 on sheep chromosome 9 (reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1), with A / C polymorphism. When the genotype of this SNP locus is AC, it has better performance in resisting Mycoplasma pneumonia than AA, and can be used as a molecular marker for the trait of sheep's resistance to Mycoplasma pneumonia.

[0018] Example 2 Detection of SNP Molecular Marker

[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 this SNP locus, specific primers for amplifying this fragment were designed:

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

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

[0022] (2) Using the specific primers in (1) to perform PCR amplification on the sheep DNA sample, an amplification product of the region containing the mutation was obtained, and the amplification product was 468bp ( 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 Novoprotein Scientific Inc., with the product number P525-02.

[0027] The amplification conditions are: using a PCR amplifier for amplification. 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, for 34 cycles; the third stage: final extension at 72°C for 5 min, and preservation at 4°C.

[0028] (3) Perform sanger sequencing on the amplification product to obtain the genotype of the SNP locus.

[0029] Example 3 Verification of SNP Molecular Marker

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

[0031] Table 2 Genotyping results of different genotypes

[0032]

[0033] The CT values of Mycoplasma pneumoniae detection in all individuals (where CT values exceeding 32 are negative) were compared. The CT value reflects the copy number of the Mycoplasma pneumoniae genome in sheep.

[0034] The comparison results are as Figure 3 shown. The heterozygous individuals containing this molecular marker had higher CT values than the wild-type individuals, that is, the copy number of the Mycoplasma pneumoniae genome was lower, with a significant difference, indicating that the individuals with genotype AC had a low load of Mycoplasma pneumoniae in sheep, and these individuals had a stronger ability to resist Mycoplasma pneumoniae.

Claims

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

1. Individuals with genotype AC at this site had higher resistance to mycoplasma pneumonia than individuals with genotype AA. The sheep breed was Hu sheep.

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 the sheep, located at position 273 of the nucleotide sequence described in SEQ ID No.1, and has an A / C polymorphism. Individuals with an AC genotype at this site have higher anti-mycoplasma pneumonia performance than individuals with an AA genotype, and the sheep breed is Hu sheep.

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.

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