SNP Molecular Markers Related to the Adaptability of Sheep to Dry and Wet Environments and Their Applications

Abstract

The present invention discloses an SNP molecular marker related to the adaptability of sheep to dry and wet environments and its application, belonging to the field of genetic engineering. The SNP locus of the SNP molecular marker is located at position 29526684 on chromosome 10 of sheep, with A / G polymorphism. When the genotype of this locus is AA, it is a sheep with adaptability to dry environments; when the genotype of this locus is GG, it is a sheep with adaptability to wet environments. It provides a new molecular breeding marker for screening or introducing sheep with adaptability to wet environments.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to an SNP molecular marker related to the adaptability of sheep to dry and wet environments and its application. Background Art

[0002] The large-scale and intensive breeding mode of meat sheep has become an inevitable trend in the development of China's meat sheep industry. In modern intensive sheep breeding production, the breeding density is increasing continuously, and the impact of environmental stress on the production and health of sheep cannot be ignored. Among them, the thermal environment is often one of the important factors affecting the development of the sheep industry. In the thermal environment, temperature and humidity are the two most sensitive factors. In the high-temperature and high-humidity environment in summer, meat sheep are extremely prone to heat stress reactions, causing physiological and biochemical changes, immune responses and metabolic disorders in meat sheep, thereby affecting the production performance of meat sheep, including feed intake, daily weight gain, slaughter time, reproductive performance and body immune function, etc., bringing huge economic losses to the summer production of the meat sheep industry. The sweat glands of sheep breeds are not well-developed, and coupled with their thick and long wool on the outside, the hot and humid climate in summer is more harmful to sheep. How to improve the production performance of sheep under summer heat stress has become a current research focus. Accelerating the cultivation of stress-resistant sheep breeds that are resistant to high temperature and high humidity is an important way to solve the above problems.

[0003] Meat sheep heat stress belongs to the category of stress. THI (Temperature-humidity index) is the most commonly used animal heat stress evaluation index, and THI 25.6 is considered extremely severe heat stress. At present, people have achieved some effects by improving environmental management, feeding management and nutritional regulation to relieve the heat stress of meat sheep, and improved the growth and reproductive performance of meat sheep in summer. However, to fundamentally solve the problem, it is still necessary to cultivate meat sheep breeds or strains that are resistant to high temperature and high humidity. The heritability of heat tolerance in domestic animals is about 0.15 - 0.30. By selecting heat-tolerant meat sheep for breeding, certain genetic progress can be obtained.

[0004] The relaxin family peptide receptor 2 (RXFP2) gene is a relaxin / insulin-like peptide receptor and is a member of the G protein-coupled family. It can be mediated by G proteins, leading to an increase in adenylate cyclase activity and an increase in cAMP. It is specifically expressed in animal embryos, testicular tissues, and epididymal tissues after birth, affecting testicular descent in male animals. Related mutations can lead to cryptorchidism. During animal embryogenesis, RXFP2 can also affect animal neural development. This gene is also the specific receptor of insulin-like factor 3 (INSL-3) in the body. The sheep RXFP2 gene is located on chromosome 10. The RXFP2 gene in sheep was obtained through genetic mapping and is considered the major gene for the presence or absence of horns and horn type in sheep. An insertion sequence of about 1.8 kb in the 3'-UTR region of the RXFP2 gene results in hornless sheep, but the specific downstream regulatory mechanism is still unclear. Researchers also found that this gene is mainly highly expressed in horn tissues and testicular tissues. Further research found that the expression level of this gene changes under different climatic environments or different breeding seasons. The RXFP2 gene can tightly bind to its ligand INSL-3 and carry out feedback regulation according to signals from the external environment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an SNP molecular marker related to the adaptability of sheep to humid environments and its application.

[0006] The technical solution of the present invention is as follows: A method for screening sheep with adaptability to humid environments, detecting the genotype of the 29,526,684th site on chromosome 10 of sheep, with the reference genome version being ARS-UI Rammb v2.0, and screening through the genotype of this site:

[0007] When the genotype of this site is AA, it is a sheep with adaptability to dry environments;

[0008] When the genotype of this site is GG, it is a sheep with adaptability to humid environments.

[0009] Application of a substance for detecting the genotype of an SNP site in the assisted breeding of sheep for dry and wet environment adaptability. The SNP site is located at the 29,526,684th position on chromosome 10 of sheep, is located at the 252nd position of the nucleotide sequence shown in SEQ ID No.1, and has A / G polymorphism.

[0010] 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.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention provides an SNP molecular marker related to the adaptability of sheep to humid environments, which is located in the intron region of the RXFP2 gene. The gene mutation at this locus significantly affects sheep. When the genotype at this locus is AA, it represents sheep with adaptability to dry environments; when the genotype at this locus is GG, it represents sheep with adaptability to humid environments. It provides a new molecular breeding marker for screening or introducing sheep with adaptability to humid environments. Description of the Drawings

[0013] Figure 1 : The degree of association between different environmental factors and the population selection signal matrix and the heterozygosity matrix;

[0014] Figure 2 : A, Local Manhattan plot of LFMM results of bio14 environmental factor on chr10, and the red dots represent the loci within the RXFP2 gene interval; B, Haplotypes of high-bio14 and low-bio14 populations in the RXFP2 gene region. Detailed Implementation Modes

[0015] Unless otherwise specified, 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.

[0016] Based on the resequencing data and climate data of sheep population samples in different humidity environments across the country, the genetic regulation of the change in sheep environmental adaptability was explored.

[0017] 1) Association analysis of environmental factors with population selection signals and heterozygosity

[0018] Different local sheep breeds were used, including the following samples:

[0019] Populations in dry environments include: Tibetan sheep PT, OL, Large Tail Han sheep, Small Tail Han sheep, Sishui Fur sheep, Wadi sheep, Tan sheep.

[0020] Populations in humid environments include: Hu sheep populations from 8 core breeding farms.

[0021] We performed redundancy analysis (RDA) on the climate data and the resequenced genomic data and found that for different genotypes, the annual temperature range, the average temperature in the warmest season, and the precipitation in the wettest month can explain most of the environmental factor variations. At the same time, it was also found that the precipitation in the driest month (bio14) was significantly associated with the selection signal values and population heterozygosity among different sheep populations ( Figure 1 ).

[0022] 2) Identification of marker loci by association analysis

[0023] Using the Latent Factor Mixed Models (LFMM), the environmental factor bio14 was associated with genomic data. The results showed that the associated loci with bio14 were mainly distributed on chromosome 6 and chromosome 10, indicating that there are major genes for the adaptation to the bio14 environmental factor. Figure 2 ) There are two alleles, A and G, at the intron region chr10:29526684 of the RXFP2 gene (the reference genome version is ARS-UI Rammb v2.0, located at position 252 of the nucleotide sequence shown in SEQ ID No.1). The frequency of the G allele is 0.58 in the general dry environment population, 0 in the OL, PT and other populations in the severe dry environment, and 0.99 in the high humidity environment population. Similarly, the change in the allele frequency of chr10:29526684 is consistent with the distribution gradient of bio14, indicating that the A allele at this locus helps sheep adapt to the dry environment, and the G allele helps sheep adapt to the humid environment. The mutation at this locus helps sheep adapt to different precipitation in different regions. Figure 2 ) It can be used as a molecular breeding marker for screening or introducing sheep with the ability to adapt to the humid environment.

[0024] 3) Genotype detection at the locus

[0025] For the Chr 10_29526684 locus, the following primers were used for PCR amplification and sequencing:

[0026] RXFP2_HF: 5’-AGGTGTTCAGTGAGCTTCCT (SEQ ID No.2)

[0027] RXFP2_HR: 5’-AGACTTCTTTTCTCTTGTTGCCT (SEQ ID No.3)

[0028] The high-fidelity PCR enzyme (2× Max MasterMix) of Nanjing Novoprotein Biotechnology Co., Ltd. was used for amplification, and the product number is P525-02.

[0029] Amplification system for Chr 10_29526684

[0030]

[0031] The amplification conditions are as follows: Amplification is carried out using a PCR amplifier. The first stage: pre-denaturation at 95°C for 5 min; the second stage: denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 45 s, for 34 cycles; the third stage: final extension at 72°C for 5 min, and preservation at 4°C. The length of the amplification product is 101 bp.

[0032] The product obtained from the previous PCR amplification is used for Sanger sequencing. The sequencing is performed by Shanghai Bioengineering Co., Ltd. After comparing the sequencing results with the reference gene sequence, the genotypes at different loci are recorded.

Claims

1. A method for screening sheep with adaptability to humid environments, characterized in that: The genotype of the 29526684th site of chromosome 10 of sheep was tested. The reference genome version was ARS-UI Rammb v2.

0. The genotype of this site was used for screening: When the genotype of this site is AA, the sheep are adaptable to dry environments; When the genotype of this site is GG, the sheep are adaptable to humid environments.

2. Application of a material for detecting the genotype of a SNP site in assisting breeding for adaptability to dry and wet environments of sheep, wherein the SNP site is located at position 29526684 of chromosome 10 of sheep, the reference genome version is ARS-UI Rammb v2.0, located at position 252 of the nucleotide sequence shown in SEQ ID No.1, and has an A / G polymorphism. When the genotype of the site is AA, the sheep is adaptable to a dry environment; when the genotype of the site is GG, the sheep is adaptable to a humid environment.

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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