Sheep KAL1 Gene SNP Molecular Marker and Its Application in Sheep Breeding

Abstract

The present invention discloses a SNP molecular marker of the sheep KAL1 gene and its application in sheep breeding, belonging to the field of molecular biology. The genotype at the 5,772,232nd position of the X chromosome of sheep is detected, and the genome version is Oar_v3.1, GCF_000298735.1. The base at this site has G / A polymorphism. When the genotype at this site changes from GG to AA, 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 particularly relates to a SNP molecular marker of a sheep KAL1 gene and its application in sheep breeding. Background Art

[0002] China is a large sheep-raising country, but the single yield level of meat sheep is low, and there is a lack of commercial meat sheep breeds with high growth performance. In meat sheep breeding, improving the growth performance of sheep has great economic benefits. The growth performance of sheep is related to multiple major genes, and base mutations in the major genes may affect the function of the genes, thereby affecting the growth performance of individuals. If molecular markers related to sheep growth traits are applied to meat sheep breeding work, more molecular markers related to growth traits need to be excavated and identified for breeding. However, there are currently few molecular markers suitable for breeding domestic local sheep breeds in China.

[0003] KAL1 (Kallmann syndrome 1, KAL1) is located on the short arm of the X chromosome and is closely related to human Kallmann syndrome (KS). The clinical symptoms of this disease are reduced secretion of gonadotropin-releasing hormone, hypogonadism and loss of smell (Stamou et al., 2018). There are also a large number of "milder" reproductive diseases related to the hypothalamic-pituitary-gonadal (HPG) axis, such as delayed puberty (Zhu et al., 2015), adult hypogonadism with low-density nutrients (Dwyer et al., 2010). Mutations in this gene not only cause defects in functions related to the gonadal axis, but also affect the development of various organs and body parts, such as the kidneys, teeth, hearing, cleft lip, etc. (Hardelin et al., 2008). Therefore, the function of this gene can regulate the growth and development of animals and is a potential molecular marker locus related to growth and development. However, whether this gene can be used as a molecular marker for breeding in sheep has not been reported yet. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a SNP molecular marker and a corresponding detection method that can be used to improve the growth rate of Chinese local sheep, and can be used to breed new sheep strains with higher growth performance.

[0005] The technical solution of the present invention is: a method for breeding sheep with high growth performance, which detects the genotype at position 5772232 of the X chromosome of sheep. The genome version is Oar_v3.1, GCF_000298735.1. The base at this site has G / A polymorphism. When the genotype at this site changes from GG to AA, it has higher growth performance.

[0006] Application of a substance for detecting the genotype of SNP locus in the assisted breeding of sheep growth performance, wherein the SNP locus is located at position 5772232 on the X chromosome of sheep, located at position 214 of the nucleotide sequence described in SEQ ID No.1, and has G / A 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] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The present invention genotypes 567 Hu sheep individuals with body weight records using a sheep 40K chip, combines the known pedigree information and phenotypic information, and uses statistical methods to conduct association analysis on growth phenotypic indicators such as body weight at different growth stages to determine the correlation between this SNP molecular marker and growth phenotypes. By designing primers to amplify the sequence containing this locus, genotype detection is performed on a certain number of sheep individuals, and the growth performance of sheep with different genotypes is compared to verify that the mutation at this locus has an impact on the growth performance of sheep 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 the comparison of the body weights of Hu sheep (AA) containing this molecular marker and Hu sheep individuals (GG) not containing this molecular marker at different months of age, and 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 SNP Molecular Marker of KAL1 Gene

[0015] Genotyping of 567 Hu sheep individuals with body weight records was performed using a 40K sheep chip. Combining the known pedigree information and phenotypic information, statistical methods were used to conduct association analysis on growth phenotypic indicators such as body weight at different growth stages. It was found that the 5,772,232nd position on the X chromosome of sheep is an SNP locus, and the base at this locus has G / A polymorphism (genome version is Oar_v3.1, GCF_000298735.1). This SNP locus is related to the growth phenotype of sheep. Compared with the wild-type GG, when the genotype at this locus mutates to AA, it has higher growth performance and can be used as a molecular marker for sheep growth traits.

[0016] Example 2 Detection of SNP Molecular Marker

[0017] (1) Based on the upstream and downstream sequences (SEQ ID No.1) of this SNP locus, specific primers for amplifying this fragment were designed:

[0018] Forward primer KAL1-F1: 5′-CAGAAGCAAATGGTGTGGGAC-3′

[0019] Reverse primer KAL1-R1: 5′-GGACAATTGGGGTACCCGTGT-3′

[0020] (2) Using the specific primers in (1) to perform PCR amplification on sheep DNA samples to obtain an amplification product of the region containing the mutation. The amplification product is 656 bp ( Figure 1 ).

[0021] The amplification system is as follows:

[0022] Table 1 KAL1 Amplification System

[0023]

[0024] Max Master Mix is a high-fidelity PCR enzyme from Nanjing Novoprotein Scientific Inc., with the product number P525-02.

[0025] The amplification conditions are as follows: Amplification is performed using a PCR amplifier. 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 preservation at 4°C.

[0026] (3) Perform sanger sequencing on the amplification product ( Figure 2 ) to obtain the genotype of the SNP locus.

[0027] Example 3 Verification of SNP Molecular Marker

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

[0029] Table 2 Weights and genotypes of 247 sheep

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] As shown in Table 2, 27 sheep were homozygous individuals containing the SNP locus mutation (genotype AA), 103 were wild-type individuals (genotype consistent with the reference genome, GG), and the rest were heterozygous individuals. The birth weights and weights at different months of age of all homozygous genotype 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 had 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 5772232 of chromosome X of sheep was detected, the genome version was Oar_v3.1, GCF_000298735.1, the base of the position had G / A polymorphism, when the genotype of the position mutated from GG to AA, the sheep 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 5772232 of the sheep X chromosome, located at position 214 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 a higher growth performance, wherein 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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