SNP molecular marker of sheep WT1 gene and its application

Abstract

The invention discloses a sheep WT1 gene SNP molecular marker and its application, belonging to the field of molecular biology, and detects the genotype of the 61369364th position of chromosome 15 of sheep. The genome version is Oar_v3.1, GCF_000298735.1, and the base of the site has G / A polymorphism. When the genotype of the site mutates from GG to AA, it has higher growth performance. Therefore, the SNP can be used as a molecular marker for sheep growth traits.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and specifically relates to a sheep WT1 gene SNP molecular marker and its application in sheep breeding. Background Art

[0002] Sheep provide a source of protein for humans. In sheep breeding, the economic traits of sheep have been continuously improved through selection. Growth traits are one of the most important indicators for the selection of meat sheep breeds. Breeding sheep strains with fast growth and larger body size is conducive to improving production efficiency and economic benefits. Molecular marker breeding can speed up breeding speed and efficiency, but there are currently few molecular markers suitable for selecting the growth rate of domestic sheep breeds.

[0003] WT1 (WT1 transcription factor, WT1) is a sequence-specific transcriptional regulator that not only helps protect cells from apoptosis (Shandilya et al., 2015), but also inhibits cell growth and differentiation by regulating the function of the spindle / mitotic node during cell division (Yao et al., 2021). Certain functional mutations in the WT1 gene can activate the cell cycle and promote cell proliferation. Therefore, the function of this gene can regulate the growth and development of animals and is a potential molecular marker site related to growth and development. However, whether this gene can be used as a molecular marker for breeding in sheep has not been reported. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a SNP molecular marker related to sheep growth traits and to provide a new molecular marker site for sheep growth trait determination.

[0005] The technical solution of the present invention is: a method for breeding sheep with high growth performance, detecting the genotype of position 61369364 of chromosome 15 of sheep, the genome version is Oar_v3.1, GCF_000298735.1, the base of the site has G / A polymorphism, and when the genotype of the site mutates from GG to AA, it has higher growth performance.

[0006] The invention discloses an application of a material for detecting the genotype of a SNP site in sheep growth performance assisted breeding, wherein the SNP site is located at position 61369364 of chromosome 15 of sheep, located at position 220 of the nucleotide sequence described in SEQ ID No. 1, and has a G / A 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] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The present invention uses sheep 40K chip to perform genotyping on 567 Hu sheep individuals with weight records, combines known pedigree information and phenotypic information, and uses statistical methods to perform association analysis on growth phenotypic indicators such as weight at different growth stages. It is found that there is a SNP site on the WT1 gene that is significantly correlated with the growth phenotype. The SNP site is located at position 61369364 of chromosome 15 of sheep (reference genome version Oar_v3.1, GCF_000298735.1), and the base of this site has a G / A polymorphism. When the genotype of this site mutates from wild-type GG to AA, the growth performance of individual sheep is significantly improved. Therefore, this SNP can be used as a molecular marker for sheep growth traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is the gel electrophoresis diagram of the PCR amplification product containing the target SNP site;

[0011] Figure 2 The sanger sequencing results of the PCR amplified product fragment;

[0012] Figure 3 The weights of Hu sheep individuals (AA) containing the molecular marker and those (GG) not containing the molecular marker at different ages were compared. The weights of mutant individuals at different ages were higher than those of wild-type individuals. DETAILED DESCRIPTION

[0013] 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.

[0014] Example 1 Discovery of WT1 gene SNP molecular markers

[0015] The present invention uses sheep 40K chip to perform genotyping on 567 Hu sheep individuals with weight records, combines known pedigree information and phenotypic information, and uses statistical methods to perform association analysis on growth phenotypic indicators such as weight at different growth stages. It is found that there is a SNP site on the WT1 gene that is significantly correlated with the growth phenotype. The SNP site is located at position 61369364 of chromosome 15 of sheep (reference genome version Oar_v3.1, GCF_000298735.1), and the base of this site has a G / A polymorphism. When the genotype of this site mutates from wild-type GG to AA, the growth performance of individual sheep is significantly improved. Therefore, this SNP can be used as a molecular marker for sheep growth traits.

[0016] Example 2 Detection of SNP Molecular Markers

[0017] (1) Based on the upstream and downstream sequences of the SNP site, the SNP is located at position 220 of the nucleotide sequence shown in SEQ ID No. 1, and specific primers for amplifying the fragment were designed:

[0018] Upstream primer WT1F1: 5′-GTCTCCTGTGATTACTGTCACC-3′

[0019] Downstream primer WT1R1: 5′-AGTCTAAGTCTTCAGGGACG-3′

[0020] (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 420 bp ( Figure 1 ).

[0021] The amplification system is as follows:

[0022] Table 1JAK2 amplification system

[0023]

[0024] 2× Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0025] The amplification conditions were as follows: amplification was performed using a PCR amplification instrument, the first stage: pre-denaturation at 95°C for 3 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, 35 cycles; the third stage: final extension at 72°C for 5 min, and storage at 4°C.

[0026] (3) The amplified product was subjected to Sanger sequencing ( Figure 2 ) to obtain the genotype of the SNP site.

[0027] Example 3 Validation of SNP Molecular Markers

[0028] The genomes of 247 sheep (the breed was Hu sheep, and the weights at different growth stages were shown in Table 2) were amplified. The amplification primers, conditions, and system were the same as those in Example 2. The genotypes of the PCR products after sequencing are shown in Table 2.

[0029] Table 2 Body weight and genotype of 247 sheep

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] The results are shown in Table 2. 78 sheep were homozygous individuals (genotype was AA) containing the SNP site mutation, 54 were wild-type individuals (genotype was consistent with the reference genome, GG type), and the rest were heterozygous individuals. The birth weight and weight at different ages of all homozygous genotype individuals were compared, and the growth curve was drawn with the mean ± standard deviation of the weight of individuals with the same genotype ( Figure 3 ), it can be seen that the homozygous mutant individuals containing this molecular marker have higher body weight at different ages than wild-type individuals.

Claims

1. A method for breeding sheep with high growth performance, characterized in that: The genotype of position 61369364 of chromosome 15 of sheep was detected, the genome version was Oar_v3.1, GCF_000298735.1, the base of this site had G / A polymorphism, and when the genotype of this site mutated from GG to AA, it had higher growth performance. The sheep breed was Hu sheep.

2. Application of a substance for detecting the genotype of a SNP site in sheep growth performance assisted breeding, wherein the SNP site is located at position 61369364 of chromosome 15 of the sheep, located at position 220 of the nucleotide sequence described in SEQ ID No.1, and has a G / A polymorphism. When the genotype of the site mutates from GG to AA, it has higher growth performance. The sheep breed is Hu sheep, and 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.

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