SNP (Single Nucleotide Polymorphism) marker related to heat stress resistance character of sheep and application of SNP marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker related to the heat stress resistance character of sheep and application of the SNP marker. According to the invention, it is found for the first time that an SNP on the PDLIM5 gene is significantly related to the heat stress resistance of sheep, and the SNP can be used as a molecular marker for the heat stress resistance of sheep. The sheep individuals identified by the method have relatively strong heat stress resistance, and can be used for breeding new sheep strains with heat stress resistance.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a SNP marker related to the heat stress resistance trait of sheep and an application thereof. Background Art

[0002] In modern intensive sheep production, the stocking density continues to increase, and the impact of environmental stress on sheep production and health cannot be ignored. Among them, the thermal environment is often one of the important factors affecting the development of the sheep farming industry. Therefore, improving the heat stress resistance of sheep is of great significance for sheep farming. Breeding sheep strains that are resistant to heat stress can improve the stress resistance of the sheep population. In sheep breeding, molecular markers with heat stress resistance can be used to identify individuals with better heat stress resistance for breeding, but there are currently few molecular markers suitable for the selection of heat stress resistance traits in domestic local sheep breeds.

[0003] PDLIM5 (PDZ and LIM domain protein 5) is also called ENH (Enigma homolog). This protein is encoded by the PDLIM5 gene. The PDLIM5 protein first discovered by Kuroda et al. is composed of one N-terminal PDZ domain and three C-terminal LIM domains, and its composition is similar to that of the Enigma protein. It was also found that the PDLIM5 gene can be found in skeletal muscle, spinal cord, etc., and its expression level is higher in skeletal muscle. The protein encoded by the PDLIM5 gene is involved in cytoskeleton assembly, cell lineage specification, organ development and tumorigenesis. The PDLIM5 gene has been found to encode multiple transcript variants of different types. The PDLIM5 gene can act as a signal regulator to affect organ development. Four splicing forms of the PDLIM5 gene have been reported in mice, including the longest subtype ENH1, whose sequence contains an NH2-terminal PDZ domain and three COOH-terminal LIM domains, and three other ENHs that do not contain LIM domains. Studies have found that ENH1 is expressed in a variety of tissues, such as the heart, brain, spleen, liver and kidney, while shorter splice forms of ENH are specifically expressed in the heart and skeletal muscle. This is different in chickens. The PDLIM5 gene is currently reported to have only one splice form, which contains a PDZ domain at the N-terminus and three LIM domains at the C-terminus. Nakagawa et al. showed that ENH co-localizes with α-actinin on the Z line. Kuroda et al. found that the PDLIM5 gene can interact with the PRKCB1 gene. The PDLIM5 gene plays an important role in promoting the differentiation of C2C12 myoblasts. Summary of the invention

[0004] The purpose of the present invention is to provide a SNP marker related to the heat stress resistance trait of sheep and application thereof.

[0005] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a SNP marker associated with the heat stress resistance trait of sheep, wherein the SNP marker contains PDLIM5 The gene has a nucleotide sequence with a polymorphism of G / A at position 366 as shown in SEQ ID NO: 1.

[0006] Furthermore, sheep individuals with the genotype AA at the polymorphic site have higher heat stress resistance than sheep individuals with the genotype GA or GG.

[0007] In the present invention, sheep PDLIM5 The reference sequence number of the gene in NCBI is NC_056059.1 ARS-UI_Ramb_v2.0.

[0008] In a second aspect, the present invention provides primers for amplifying the SNP marker, comprising an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3.

[0009] In a third aspect, the present invention provides a detection reagent or a kit containing the primers.

[0010] In a fourth aspect, the present invention provides a method for identifying and breeding a heat stress resistant sheep strain, comprising: 1) Extract the total DNA of the sheep to be tested; 2) using DNA as a template and performing PCR amplification using primers shown in SEQ ID NO: 2-3; 3) Analyze the PCR amplification products.

[0011] Preferably, the PCR reaction system is: 2 × Phanta Max Master Mix 12.5 μL, DNA template 50-100 ng, 10 μM upstream and downstream primers 1 μL each, and ddH2O supplemented to 25 μL.

[0012] Preferably, the PCR reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; final extension at 72°C for 5 min.

[0013] Furthermore, step 3) includes: performing Sanger sequencing on the amplified product to obtain the genotype of the site with the polymorphism, and sheep individuals with genotype AA have higher heat stress resistance than sheep individuals with genotype GA and GG.

[0014] In a fifth aspect, the present invention provides any of the following applications of the SNP marker or a detection reagent thereof: (1) Used for early prediction of heat stress resistance in sheep; (2) Used for molecular marker-assisted breeding of sheep.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention was first discovered in PDLIM5 There is a SNP on the gene that is significantly associated with the sheep's heat stress resistance trait and can be used as a molecular marker for the sheep's heat stress resistance trait. The sheep individuals identified by this method have relatively strong heat stress resistance and can be used to breed new sheep strains with heat stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a significant signal site related to sheep humidity adaptability obtained through gene association analysis in a preferred embodiment of the present invention.

[0017] Figure 2 This is a PCR result diagram of the target SNP site in a preferred embodiment of the present invention. The leftmost band is the DNA Marker, and the remaining bands are PCR amplification bands of sheep DNA.

[0018] Figure 3 This is the Sanger sequencing result of the PCR product fragment in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0019] The present invention aims to provide a molecular marker site and a corresponding detection method that can be used to screen heat stress resistance in Chinese local sheep.

[0020] The present invention also provides a PDLIM5 Gene molecular markers and their application in sheep breeding.

[0021] The present invention adopts the following technical solution: The invention provides a method for breeding sheep with high heat stress resistance traits. The genotype of position 30590931 of chromosome 6 of sheep is detected, and the reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1. Individuals with genotype AA at the site have higher heat stress resistance than individuals with genotypes GG and GA.

[0022] The present invention also provides the use of a material for detecting the genotype of a SNP site in auxiliary breeding of sheep heat stress resistance traits, wherein the SNP site is located at position 30590931 of chromosome 6 of sheep, located at position 366 of the nucleotide sequence described in SEQ ID NO: 1, and has a G / A polymorphism (n=g or a).

[0023] 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.

[0024] The present invention also provides a primer pair, the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3.

[0025] The invention also provides a kit, which contains the primer pair.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] Example 1 PDLIM5 Discovery of gene SNP molecular markers By performing redundancy analysis (RDA) on climate data and resequencing genomic data, it was found that for different genotypes, the annual temperature difference (bio7) and the precipitation in the driest month (bio14) can explain most of the variation in environmental factors. Resequencing data of 400 individuals of 8 local sheep breeds (Tibetan sheep, Oula sheep, large-tailed Han sheep, small-tailed Han sheep, Sishui fur sheep, depression sheep, beach sheep, and lake sheep) living in different temperature areas were used. Latent Factor Mixed Models (LFMM) were used to perform association analysis between the environmental factor bio14 and the genomic data. The results showed that the significant sites associated with bio14 were mainly distributed on chromosomes 6 and 10. This suggests that there are major effect genes ( Figure 1 ).in PDLIM5 There are two alleles, G and A, at the chr6:30590931 site in the intron region of the gene (the reference genome version is ARS-UI Rammb v2.0). The gene frequency of the A allele is significantly higher than that of the G allele in the sheep population that can tolerate high temperature and humidity; the gene frequency of the G allele is significantly higher than that of the A allele in the sheep population that cannot tolerate high temperature and humidity. Similarly, the change in the frequency of the chr6: 30590931 allele is consistent with the distribution gradient of bio14, suggesting that the A allele at this site helps sheep adapt to high humidity environments and resist heat stress ( Figure 1 ), which can be used as a molecular breeding marker for screening or introducing sheep with heat stress adaptability.

[0028] exist PDLIM5There is a SNP on the gene that is significantly correlated with the sheep's resistance to heat stress. The SNP is located at position 30590931 on chromosome 6 of the sheep (the reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1) and has a G / A polymorphism. When the genotype of this SNP site carries the A allele, it has better resistance to high humidity than carrying the G allele, and can be used as a molecular marker for the sheep's resistance to high humidity.

[0029] Example 2 Detection of SNP Molecular Markers The detection method of SNP molecular markers provided in this embodiment includes: (1) The SNP is located at position 366 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: Upstream primer PDLIM5F1: 5′-TCTTAGCCTGGGATAAGGT -3′ (SEQ ID NO: 2) Downstream primer PDLIM5R1: 5′-TCCAGGTAGACCACATGGCA -3′ (SEQ ID NO: 3) (2) PCR amplification was performed on the sheep DNA sample using the specific primers in (1) to obtain an amplification product of the region containing the mutation. The amplification product was 575 bp ( Figure 2). The sequence is as follows (the 366th base is the mutation sequence), which is a G>A mutation: 5'--3' (SEQ ID NO: 1).

[0030] The amplification system is shown in Table 1: Table 1 PDLIM5 Amplification system

[0031] 2 × Phanta® Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0032] 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 60°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.

[0033] (3) Perform Sanger sequencing on the amplified product to obtain the genotype of the SNP site ( Figure 3 ).

[0034] Example 3 Verification of SNP Molecular Markers The genomes of 78 sheep (Hu sheep breed) were amplified, and the amplification primers, amplification conditions, and amplification system were the same as in Example 2. The results of PCR product sequencing showed that 29 sheep were homozygous individuals (genotype was AA) containing the SNP site, 35 sheep were heterozygous individuals (genotype was GA) containing the SNP site, and 14 were wild-type individuals (genotype was GG, consistent with the reference genome).

[0035] The respiratory rates (times / minute) of all individuals were compared, where the respiratory rate reflected the degree of heat stress.

[0036] The comparison results are shown in Table 2. The homozygous individuals of the mutation containing this molecular marker have a lower respiratory frequency than heterozygous individuals and wild-type individuals, that is, the degree of heat stress is lower, which is a significant difference, indicating that sheep individuals with genotype AA have a lower respiratory frequency and are more resistant to heat stress.

[0037] Table 2 Analysis of variance of average respiratory rate of sheep with different genotypes Note: Different lowercase letters in the same group indicate significant differences (P<0.05).

[0038] From the above experimental results, it can be seen that the 30590931 site on chromosome 6 of sheep (the reference genome version is ARS-UI_Ramb_v2.0, GCF_016772045.1) has a G / A polymorphism. When the genotype of this site is AA, the average respiratory rate and heat stress indicators are significantly reduced. Sheep individuals with genotype AA have better resistance to heat stress than GA and GG individuals.

[0039] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A SNP marker associated with heat stress resistance in sheep, characterized in that: The SNP marker contains PDLIM5 The gene has a nucleotide sequence with a polymorphism of G / A at position 366 as shown in SEQ ID NO:

1.

2. The SNP marker according to claim 1, characterized in that Sheep individuals with the AA genotype at the polymorphic site have higher heat stress resistance than sheep individuals with the GA and GG genotypes.

3. A primer for amplifying the SNP marker according to claim 1 or 2, characterized in that: It includes an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:

3.

4. A detection reagent or kit containing the primers according to claim 3.

5. A method for identifying and breeding heat stress resistant sheep strains, characterized in that: include: 1) Extract the total DNA of the sheep to be tested; 2) using DNA as a template and performing PCR amplification using primers shown in SEQ ID NO: 2-3; 3) Analyze the PCR amplification products.

6. The method according to claim 5, characterized in that The PCR reaction system was: 2 × Phanta Max MasterMix 12.5 μL, DNA template 50-100 ng, 1 μL each of 10 μM upstream and downstream primers, and ddH2O supplemented to 25 μL.

7. The method according to claim 5, characterized in that The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; and final extension at 72°C for 5 min.

8. The method according to claim 5 or 6, characterized in that: Step 3) includes: performing Sanger sequencing on the amplified product to obtain the genotype of the site with the polymorphism, and sheep individuals with genotype AA have higher heat stress resistance than sheep individuals with genotype GA and GG.

9. Any of the following uses of the SNP marker or detection reagent thereof according to claim 1 or 2: (1) Early prediction of heat stress resistance in sheep; (2) Used for molecular marker-assisted breeding of sheep.

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