Sheep PLCL1 Gene SNP Molecular Marker and Its Application

Abstract

The present invention discloses an SNP molecular marker of the sheep PLCL1 gene and its application, belonging to the field of molecular biology. The genotype at position 199225518 on chromosome 2 of sheep is detected, with the genome version being Oar_v3.1, GCF_000298735.1. The base at this site has C / T polymorphism. When the genotype at this site mutates from CC to TT, it has higher growth performance. It can be used to breed new sheep strains with higher growth performance.

Description

Technical Field

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

[0002] Sheep are one of the most important livestock. China is the largest sheep breeding and consuming country in the world. In sheep breeding, improving the growth performance of sheep can bring huge economic benefits. In order to improve the growth performance of sheep, major genes related to the growth and development of sheep can be targeted for the breeding of high-growth-performance sheep. This requires the identification and verification of molecular markers that can be used for breeding, but currently there are few molecular markers related to the growth performance of sheep.

[0003] PLCL1, also known as PRIP, is a gene related to human hip bone 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 a mouse model with knockout (KO) of the PRIP gene, an increase in bone density and trabeculae, a decrease in osteoclast differentiation ability, and a reduction in bone resorption were observed, indicating that PRIP affects the calcium-calcineurin-NFATc1 signaling pathway by regulating intracellular Ca2+ concentration, and plays an important role in bone growth and differentiation (Murakami et al., 2017). The function of this gene is related to the growth and development of animals, but there is no report 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 locus for the determination of sheep growth traits.

[0005] The technical solution of the present invention is: a method for breeding high-growth-performance sheep, detecting the genotype at position 199225518 on chromosome 2 of sheep, with the genome version of Oar_v3.1, GCF_000298735.1. The base at this site has C / T polymorphism. When the genotype at this site changes from CC to TT, it has higher growth performance.

[0006] Application of a substance for detecting the genotype of a SNP locus in the assisted breeding of sheep growth performance. The SNP locus is located at position 199225518 on chromosome 2 of sheep, located at the 106th position of the nucleotide sequence shown in SEQ ID No.1, and has C / T polymorphism.

[0007] Further, the substance is a primer pair, and the nucleotide sequences of the primer pair are 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 provides an SNP molecular marker locus that can be used for sheep molecular breeding. This locus is located on the PLCL1 gene and provides a method for detecting this locus. The sheep individuals identified by this method have higher body weights and can be used to breed new sheep strains with higher growth performance. Description of the Drawings

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

[0011] Figure 2 It is the sanger sequencing result of the PCR amplification product fragment;

[0012] Figure 3 It is a comparison of the body weights of Hu sheep (TT) containing this molecular marker and Hu sheep individuals (CC) not containing this molecular marker at different months of age. The body weights of mutant individuals at different months of age are higher than those of wild-type individuals. Detailed Embodiments

[0013] 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.

[0014] Example 1 Discovery of the SNP Molecular Marker of the PLCL1 Gene

[0015] Genotyping was performed on 567 Hu sheep individuals with body weight records using a sheep 40K chip. Combining the known pedigree information and phenotypic information, statistical methods were used to perform association analysis on growth phenotypic indicators such as body weight at different growth stages. It was found that there was an SNP locus on the sheep PLCL1 gene that was significantly correlated with growth phenotypes. This SNP locus is located at position 199225518 on chromosome 2 of sheep (Oar_v3.1, GCF_000298735.1). The base at this locus has C / T polymorphism. When the genotype at this locus changes from CC to TT, the growth performance of sheep individuals is significantly improved. Therefore, this SNP can be used as a molecular marker for sheep growth traits.

[0016] Example 2 Detection of the SNP Molecular Marker

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

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

[0019] Reverse primer PLCL1-R1: 5′-TGCCAGTGTGTTTGCTTCTCAG-3′

[0020] (2) Use the specific primers in (1) to perform PCR amplification on the sheep DNA sample to obtain an amplification product of the region containing the mutation. The amplification product is 441 bp ( Figure 1 ).

[0021] The amplification system is as follows:

[0022] Table 1 JAK2 amplification system

[0023]

[0024]

[0025] 2× Max Master Mix is a high-fidelity PCR enzyme from Nanjing Novoprotein Scientific Co., Ltd., with the product number P525-02.

[0026] The amplification conditions are as follows: Use a PCR amplifier for amplification. 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, for 35 cycles; the third stage: final extension at 72°C for 5 min, and store at 4°C.

[0027] (3) Perform Sanger sequencing on the amplification product ( Figure 2 ) to obtain the genotype of the SNP locus.

[0028] Example 3 Verification of SNP molecular markers

[0029] Amplify the genomes of 247 sheep (the breed is Hu sheep, and the weights at different growth stages are shown in Table 2). The amplification primer conditions and system are the same as in Example 2. The genotypes after sequencing of the PCR products are shown in Table 2.

[0030] Table 2 Weights and genotypes of 247 sheep

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037] The results are shown in Table 2. 33 sheep are homozygous individuals containing the SNP locus mutation (genotype TT), 130 are wild-type individuals (genotype is the same as the reference genome, CC), and the rest are heterozygous individuals. The birth weights and weights at different months of age of the homozygous individuals were compared, and the growth curves were plotted with the mean ± standard deviation of the weights of individuals with the same genotype ( Figure 3 ). It can be seen that the homozygous mutant individuals containing this molecular marker have higher weights at different months of age than the 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 position had C / T polymorphism, when the genotype of this position mutated from CC to TT, it had higher growth performance, and the growth performance was body weight.

2. 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 chromosome 2 of sheep, the genome version is Oar_v3.1, GCF_000298735.1, and is located at position 106 of the nucleotide sequence shown in SEQ ID No. 1, and has a C / T polymorphism. When the genotype of the site mutates from CC to TT, it has higher growth performance, and the growth performance is body weight.

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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