Sheep PLCL1 gene SNP molecular marker and application thereof

Abstract

The invention discloses a sheep PLCL1 gene SNP molecular marker and application thereof, and belongs to the field of molecular biology, the genotype of the 199225518 site of a sheep chromosome 2 is detected, the genomic version is Oarv3.1, GCF000298735.1, the base of the site has C / T polymorphism, and when the genotype of the site is mutated from CC to TT, the site has higher growth performance. The method can be used for breeding new sheep strains with higher growth performance.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a sheep PLCL1 gene SNP molecular marker and application thereof in sheep breeding. Background Art

[0002] Sheep is one of the most important livestock. my country is the world's largest sheep farmer and consumer. In sheep farming, improving the growth performance of sheep can bring huge economic benefits. In order to improve the growth performance of sheep, the main effect genes related to sheep growth and development can be used to select sheep with high growth performance. This requires the identification and verification of molecular markers that can be used for breeding, but there are currently few molecular markers related to sheep growth performance.

[0003] PLCL1, also known as PRIP, is a gene associated with human hip size (Liu et al., 2009) and may play a role by participating in the regulation of the Ca2+ signaling pathway (Zofková, 2011; Wu et al., 2020). In the PRIP knockout (KO) mouse model, an increase in bone density and trabecular bone, a decrease in osteoclast differentiation ability, and a decrease in bone resorption were observed, indicating that PRIP plays an important role in bone growth and differentiation by regulating intracellular Ca2+ concentration and then affecting the calcium-calcineurin-NFATc1 signaling pathway (Murakami et al., 2017). The function of this gene is related to the growth and development of animals, but there is a lack of reports on whether this gene can be used for molecular breeding of sheep. 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 199225518 of chromosome 2 of sheep, the genome version is Oar_v3.1, GCF_000298735.1, the base of the site has C / T polymorphism, and when the genotype of the site mutates from CC to TT, 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 199225518 of sheep chromosome 2, located at position 106 of the nucleotide sequence described in SEQ ID No. 1, and has a C / T 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 invention provides a SNP molecular marker site that can be used for sheep molecular breeding. The site is located on the PLCL1 gene and a method for detecting the site is provided. The sheep individuals identified by the method have higher body weight and can be used to breed new sheep strains with higher growth performance. 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 body weights of Hu sheep individuals (TT) containing the molecular marker and those (CC) not containing the molecular marker at different months of age were compared. The body weights of mutant individuals at different months of age were all 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 SNP Molecular Markers of PLCL1 Gene

[0015] The sheep 40K chip was used to perform genotyping on 567 Hu sheep individuals with weight records. Combined with the known pedigree information and phenotypic information, statistical methods were used to conduct association analysis on growth phenotypic indicators such as weight at different growth stages. It was found that there was a SNP site on the sheep PLCL1 gene that was significantly correlated with the growth phenotype. The SNP site was located at position 199225518 on chromosome 2 of sheep (Oar_v3.1, GCF_000298735.1). The base of this site had a C / T polymorphism. When the genotype of this site mutated from CC to TT, the growth performance of the sheep individual was 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) The SNP is located at position 106 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:

[0018] Upstream primer PLCL1-F1: 5′-GCTGGGTGATAATCAGAAACTG-3′

[0019] Downstream primer PLCL1-R1: 5′-TGCCAGTGTGTTTGCTTCTCAG-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 441 bp ( Figure 1 ).

[0021] The amplification system is as follows:

[0022] Table 1JAK2 amplification system

[0023]

[0024]

[0025] 2× Max Master Mix is ​​a high-fidelity PCR enzyme from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0026] 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 57°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.

[0027] (3) The amplified product was subjected to Sanger sequencing ( Figure 2 ) to obtain the genotype of the SNP site.

[0028] Example 3 Validation of SNP Molecular Markers

[0029] 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 primer conditions and system were the same as in Example 2. The genotypes after PCR product sequencing are shown in Table 2.

[0030] Table 2 Body weight and genotype of 247 sheep

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] The results are shown in Table 2. 33 sheep were homozygous individuals (genotype was TT) containing the SNP site mutation, 130 were wild-type individuals (genotype was consistent with the reference genome, CC), and the rest were heterozygous individuals. The birth weight and weight at different ages of individuals with homozygous genotypes 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 199225518 of chromosome 2 of sheep was detected. The genome version was Oar_v3.1, GCF_000298735.

1. The base of this site had C / T polymorphism. When the genotype of this site mutated from CC to TT, it had higher growth performance.

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 199225518 of sheep chromosome 2, located at position 106 of the nucleotide sequence described in SEQ ID No. 1, and has a C / T 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.

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