SNP (Single Nucleotide Polymorphism) molecular marker related to dry and wet environment adaptability of sheep and application thereof

Abstract

The invention discloses an SNP molecular marker related to dry and wet environment adaptability of sheep and application of the SNP molecular marker, and belongs to the field of genetic engineering, the SNP site of the SNP molecular marker is located at the 29526684th site of the 10th chromosome of the sheep, and the SNP molecular marker has A / G polymorphism. When the genotype of the site is AA, the sheep is suitable for a dry environment; and when the genotype of the site is GG, the sheep is suitable for a humid environment. A novel molecular breeding marker is provided for screening or introducing sheep adapting to a humid environment.

Description

Technical Field

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

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

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

[0004] The relaxin family peptide receptor 2 (RXFP2) gene is a relaxin / insulin-like peptide receptor and a member of the G protein-coupled family. It can be mediated by G proteins, leading to an increase in adenylate cyclase activity and cAMP. It is specifically expressed in the testicular tissue and epididymal tissue of animals during the embryonic period and after birth, affecting the testicular sedimentation of male animals. Related mutations can lead to cryptorchidism. In the embryonic period, RXFP2 can also affect the development of animal nerves. This gene is also a specific receptor for insulin-like factor 3 (INSL-3) in the body. The sheep RXFP2 gene is located on chromosome 10. The RXFP2 gene was obtained through genetic positioning in sheep and is considered to be the main effect gene for the presence or absence of horns and the type of horns in sheep. A 1.8kb insertion sequence in the 3'-UTR segment of the RXFP2 gene will cause sheep to be hornless, but the specific downstream regulatory mechanism is still unclear. The researchers also found that the gene is highly expressed mainly in horn tissues and testicular tissues. Further studies have found that the expression level of the gene changes under different climatic conditions or different breeding seasons. The RXFP2 gene and its ligand INSL-3 can bind tightly and perform 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 a SNP molecular marker related to the adaptability of sheep to humid environment and its application.

[0006] The technical solution of the present invention is: a method for screening sheep with adaptability to humid environments, detecting the genotype of the 29526684th site of chromosome 10 of sheep, the reference genome version is ARS-UI Rammb v2.0, and screening is performed based on the genotype of the site:

[0007] When the genotype of this site is AA, the sheep are adaptable to dry environments;

[0008] When the genotype of this site is GG, the sheep are adaptable to humid environments.

[0009] The invention discloses a material for detecting the genotype of a SNP site in sheep dry and wet environment adaptability auxiliary breeding, wherein the SNP site is located at position 29526684 of chromosome 10 of sheep, located at position 252 of the nucleotide sequence described in SEQ ID No. 1, and has an A / G polymorphism.

[0010] 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.

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

[0012] The present invention provides a 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 site significantly affects sheep. 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. This provides a new molecular breeding marker for screening or introducing sheep that can adapt to humid environments. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0014] Figure 2 : A, local Manhattan plot of chr10 of LFMM results of bio14 environmental factor, red dots represent sites within the RXFP2 gene region; B, haplotypes of high bio14 and low bio14 populations in the RXFP2 gene region. DETAILED DESCRIPTION

[0015] 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.

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

[0017] 1) Analysis of association between environmental factors, population selection signals and heterozygosity

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

[0019] Groups in dry environments include: Tibetan sheep PT, OL, big-tailed Han sheep, small-tailed Han sheep, Sishui fur sheep, lowland sheep, and beach sheep.

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

[0021] We conducted a redundancy analysis (RDA) on the climate data and the resequencing genome data and found that for different genotypes, the annual temperature range, the average temperature of the warmest season and the precipitation in the wettest month can explain most of the variation in environmental factors. We also found that the precipitation in the driest month (bio14) was significantly associated with the selection signal value and population heterozygosity between different sheep populations ( Figure 1 ).

[0022] 2) Association analysis to identify marker loci

[0023] Using latent factor mixed models (LFMM), we conducted an association analysis between the bio14 environmental factor and genomic data. The results showed that the bio14-associated loci were mainly distributed on chromosomes 6 and 10. This suggests that there are major effect genes ( Figure 2 ). Among them, there are two alleles, A and G, at the chr10:29526684 site in the intron region 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 in the normal dry environment population is 0.58, in the OL, PT and other populations under severe dry environments, the G allele frequency is 0, and in the high-humidity environment population, the frequency is 0.99. Similarly, the change in the frequency of the chr10:29526684 allele is consistent with the bio14 distribution gradient, suggesting that the A allele at this site helps sheep adapt to dry environments, the G allele helps sheep adapt to humid environments, and the mutation of this site helps sheep adapt to different precipitation in different regions ( Figure 2 ), which can be used as a molecular breeding marker for screening or introducing sheep with adaptability to humid environments.

[0024] 3) Site genotype detection

[0025] For Chr 10_29526684 site, PCR amplification and sequencing were performed using the following primers:

[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) for amplification, catalog number P525-02.

[0029] Chr 10_29526684 amplification system

[0030]

[0031] Amplification conditions: amplification using a PCR amplification instrument, first stage: 95℃ pre-denaturation for 5min; second stage: 95℃ denaturation for 30s, 59℃ annealing for 30s, 72℃ extension for 45s, 34 cycles; third stage: 72℃ final extension for 5min, 4℃ storage. The length of the amplified product is 101bp.

[0032] The product obtained by PCR amplification in the previous step was used for Sanger sequencing, which was performed by Shanghai Bioengineering Co., Ltd. After comparing the sequencing results with the reference gene sequence, the genotypes of different sites were 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 of sheep adaptability to dry and wet environments, wherein the SNP site is located at position 29526684 of chromosome 10 of sheep, located at position 252 of the nucleotide sequence described in SEQ ID No. 1, and has an A / G 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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