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 molecular biology. The SNP molecular marker related to the humid environment adaptability of the sheep is located in an intron region of a BMPR1B gene, the gene mutation of the site significantly affects the sheep, and when the genotype of the site is AA, the sheep is the sheep with dry environment adaptability; when the genotype of the locus is GG, the locus is a sheep adaptive to a humid environment, so that a new molecular breeding marker is provided for screening or introducing the sheep adaptive to the humid environment.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, 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] Bone morphogenetic protein receptor 1 B BMPR1B) is also known as ALK6 (Activin receptor-like kinases 6), located on chromosome 6 of sheep. It is a membrane receptor for bone morphogenetic protein and a member of the transforming growth factor-β (TGF-β) superfamily. It is widely present in various tissues of the animal body, but is mainly expressed in ovarian oocytes and granulosa cells. It plays an important role in cumulus cell expansion, ovulation cycle and skeletal system development. It is mainly involved in the signal transduction of BMP2, BMP4, BMP 6 / Vgr 1 (Vg-related protein 1), BMP7 / OP1 (Osteogenic protein 1), GDF5 and BMP 15. It is an important transmembrane receptor protein. Well-known FcB The mutation is the A746G mutation in the coding region of the sheep BMPR1B gene, which causes the amino acid at position 249 to change from glutamine to arginine (Q249R), and has been officially named by the International Committee on Genetic Nomenclature of Sheep and Goats FcB Gene, this mutation will increase the number of ovulations and the number of lambs with an additive effect. In recent years, it has been found that BMPR1B activity is regulated by the small molecule inhibitory protein FKBP1A, which plays a key role in controlling the activity of BMPR1B and BMP / SMAD pathways. BMPR1B Whether genes play a role in environmental adaptation has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide a SNP molecular marker related to the adaptability of sheep to dry and wet environments and an application thereof.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a SNP molecular marker related to the adaptability of sheep to dry and wet environments, wherein the molecular marker contains BMPR1B The gene has a nucleotide sequence with the polymorphism at position 289 being A / G as shown in SEQ ID NO: 1.

[0007] Furthermore, when the genotype of the site with the polymorphism is AA, the sheep are adaptable to dry environments; when the genotype of the site with the polymorphism is GG, the sheep are adaptable to humid environments.

[0008] In the present invention, sheep BMPR1B The reference sequence number of the gene in NCBI is NC_056059.1 ARS-UI_Ramb_v2.0.

[0009] In a second aspect, the present invention provides primers for amplifying the molecular marker, comprising an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:3.

[0010] In a third aspect, the present invention provides a detection reagent or a kit containing the primers.

[0011] In a fourth aspect, the present invention provides a method for identifying and breeding sheep strains with adaptability to humid environments, comprising: 1) Extract the total DNA of the sheep to be tested; 2) using DNA as a template and performing PCR amplification using primers shown in SEQ ID NO: 2-3; 3) Analyze the PCR amplification products.

[0012] Preferably, the PCR reaction system is: 2 × Phanta Max Master Mix 12.5 μL, DNA template 50-100 ng, 10 μM upstream and downstream primers 1 μL each, ddH 2 Add 25 μL of HO.

[0013] Preferably, the PCR reaction program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 34 cycles; final extension at 72°C for 5 min.

[0014] Furthermore, step 3) includes: performing Sanger sequencing on the amplified product to obtain the genotype of the site having the polymorphism, and when the genotype is GG, the sheep to be tested is a sheep that is adaptable to a humid environment.

[0015] In a fifth aspect, the present invention provides any of the following applications of the molecular marker or its detection reagent: (1) Used for early prediction of sheep's adaptability to dry and wet environments; (2) Used for molecular marker-assisted breeding of sheep.

[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides a SNP molecular marker related to sheep's adaptability to humid environments, located at BMPR1B In the intron region of the 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, thus providing a new molecular breeding marker for screening or introducing sheep that can adapt to humid environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1It is the correlation degree between different environmental factors and population selection signal matrix and heterozygosity matrix in the preferred embodiment of the present invention.

[0018] Figure 2 This is a preferred embodiment of the present invention, in which the BMPR1B gene interval on the sheep genome is significantly associated with the precipitation (bio14) and humidity in the driest month in the sheep's area. Among them, A, bio14 environmental factor LFMM result chr6 local Manhattan plot, red points represent BMPR1B Sites within the gene interval; B, high bio14 and low bio14 populations BMPR1B Haplotype of a gene region.

[0019] Figure 3 This is a PCR result diagram of the target SNP site in a preferred embodiment of the present invention, wherein the leftmost band is a DNA marker, and the remaining bands are PCR amplification bands of sheep DNA.

[0020] Figure 4 This is the Sanger sequencing result of the PCR product fragment in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0021] The present invention aims to provide a SNP molecular marker related to the adaptability of sheep to humid environment and application thereof.

[0022] The invention also provides a method for selecting sheep with adaptability to humid environments.

[0023] The present invention adopts the following technical solution: The present invention provides a method for screening sheep with adaptability to humid environments, wherein the genotype of the 30090758th site of chromosome 6 of sheep is detected, and the reference genome version is ARS-UI Rammb v2.0, and the genotype of the site is 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.

[0024] The present invention also provides the use of a material for detecting the genotype of a SNP site in auxiliary breeding for the adaptability of sheep to dry and wet environments. The SNP site is located at position 30090758 of chromosome 6 of sheep, located at position 289 of the nucleotide sequence described in SEQ ID NO: 1, and has an A / G polymorphism (n=a or g).

[0025] Furthermore, the substance is a primer pair, and the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3.

[0026] The present invention also provides a primer pair, the nucleotide sequence of the primer pair is shown in SEQ ID NO: 2-3.

[0027] The invention also provides a kit, which contains the primer pair.

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Example 1 BMPR1B Discovery of gene SNP molecular markers 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.

[0030] 1. Analysis of association between environmental factors, population selection signals and heterozygosity Different local sheep breeds were used, including the following samples: Groups in dry environments include: Tibetan sheep, Oula sheep, big-tailed Han sheep, small-tailed Han sheep, Sishui fur sheep, lowland sheep, and beach sheep.

[0031] The groups in humid environments include: Hu sheep groups from 8 core breeding farms.

[0032] By combining climate data with resequencing genome data, we conducted a redundancy analysis (RDA) 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 ).

[0033] 2. Identification of marker loci by association analysis 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 for adaptation to the bio14 environmental factor ( Figure 2 ).in BMPR1BIn the intron region of the gene, chr6: 30090758 (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 A allele is dominant in the dry environment population, with a frequency of 0.67, and the G allele is dominant in the high-humidity environment population, with a frequency as high as 0.97. The allele frequency is consistent with the distribution gradient trend of the BIO14 environmental factor, suggesting that the A allele at this site helps sheep adapt to dry environments, and the G allele helps sheep adapt to humid environments. 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.

[0034] 3. Site genotype detection For Chr 6_3009075 site, PCR amplification and sequencing were performed using the following primers: BMPR1B _HF: 5'-AAGCACTCCCAAGACAGCTTG (SEQ ID NO:2) BMPR1B _HR: 5'-GTCCCTGCAGTGTACGATTGA (SEQ ID NO:3) Amplification was performed using a high-fidelity PCR enzyme (2 × Phanta® MaxMaster Mix) from Nanjing Novozymes Biotech Co., Ltd., catalog number P525-02.

[0035] Chr6: 30090758 Amplification system see Table 1: Table 1 chr6: 30090758 amplification system

[0036]

[0037] The product obtained by PCR amplification in the previous step was used for Sanger sequencing ( Figure 3 ), sequencing was performed by Shanghai Bioengineering Co., Ltd. After comparing the sequencing results with the reference gene sequence, the genotypes of different SNP sites can be obtained ( Figure 4 ).

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A SNP molecular marker related to the adaptability of sheep to dry and wet environments, characterized in that: The molecular marker contains sheep BMPR1B The gene has a nucleotide sequence with the polymorphism at position 289 being A / G as shown in SEQ ID NO:

1.

2. The molecular marker according to claim 1, characterized in that When the genotype of the site with the polymorphism is AA, the sheep are adaptable to dry environments; when the genotype of the site with the polymorphism is GG, the sheep are adaptable to humid environments.

3. A primer for amplifying the molecular marker according to claim 1 or 2, characterized in that: It includes an upstream primer as shown in SEQ ID NO:2 and a downstream primer as shown in SEQ ID NO:

3.

4. A detection reagent or kit containing the primers according to claim 3.

5. A method for identifying and breeding sheep strains that are adaptable to humid environments, characterized in that: include: 1) Extract the total DNA of the sheep to be tested; 2) using DNA as a template and performing PCR amplification using primers shown in SEQ ID NO: 2-3; 3) Analyze the PCR amplification products.

6. The method according to claim 5, characterized in that The PCR reaction system was: 2 × Phanta Max MasterMix 12.5 μL, DNA template 50-100 ng, 1 μL each of 10 μM upstream and downstream primers, and ddH2O supplemented to 25 μL.

7. The method according to claim 5, characterized in that The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 60 s, 34 cycles; and final extension at 72°C for 5 min.

8. The method according to claim 5 or 6, characterized in that: Step 3) comprises: performing Sanger sequencing on the amplified product to obtain the genotype of the site having the polymorphism, and when the genotype is GG, the sheep to be tested is a sheep that is adaptable to a humid environment.

9. Any of the following uses of the molecular marker or detection reagent thereof according to claim 1 or 2: (1) Used for early prediction of sheep's adaptability to dry and wet environments; (2) Used for molecular marker-assisted breeding of sheep.

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