CN115820866A - Molecular marker, primer pair, kit and identification method related to sheep double muscle phenotype traits - Google Patents

Abstract

The invention discloses molecular markers, primer pairs, kits and identification methods related to sheep double muscle phenotype traits. A technical solution to be protected by the present invention is the application of materials for detecting the polymorphism or genotype of the SNP site in the sheep genome in the preparation of identification or auxiliary identification of sheep double-muscle phenotypic traits; the SNP site is the sheep genome A SNP site of , which is the 235th nucleotide of sequence 1 in the sequence listing, which is A or G. Experiments have proved that the relevant detection primers of the molecular marker can determine the genotype of the sheep through the PCR product according to the detection of the molecular marker in the sheep genome, can effectively identify the double-muscle phenotype traits of the sheep, and can be used for early breeding of sheep. Even when the sheep are just born, they can be screened to improve the selection efficiency of sheep breeding with double-muscle phenotype traits and speed up the breeding process.

Description

Molecular markers, primer pairs, kits and identification related to sheep double muscle phenotypic traits method

technical field

The invention relates to the field of biological technology, in particular to a molecular marker, primer pair, kit and identification method related to the phenotypic traits of sheep double muscle.

Background technique

The meat production performance of livestock is an important economic trait in animal husbandry production, and animal husbandry workers are constantly devoting themselves to the selection and breeding of varieties with high meat production and good meat quality. Traditional breeding work is time-consuming and labor-intensive, but with the rapid development of life sciences and molecular biotechnology, researchers are trying to use the combination of molecular breeding and traditional breeding methods to breed livestock breeds with obvious breakthroughs in meat production.

Studies have shown that Myostatin (MSTN) can inhibit the proliferation and differentiation of muscle cells, and negatively regulate the development and regeneration of skeletal muscle. The MSTN gene is highly conserved in mammals, and overexpression will reduce muscle weight; MSTN-deficient livestock have increased skeletal muscle weight, which is the so-called double-muscle phenotype. There are double-muscle breeds due to MSTN gene mutations and long-term selection in nature. Such as Piedmont cattle, Belgian blue cattle. About 20 different types of genetic variation have been identified in mammalian MSTN genes, including deletions, insertions, and single nucleotide polymorphisms. MSTN-deficient and suppressed livestock have increased skeletal muscle weight and reduced fat content, which greatly increases the ratio of muscle to other tissues.

Because the mutation of MSTN gene significantly improves the meat production performance of livestock, this gene has become a hotspot gene in the field of animal molecular breeding, and it is of great significance to selectively breed double-muscle sheep with MSTN mutation.

Contents of the invention

The technical problem to be solved by the invention is how to detect the double-muscle phenotype character of sheep or how to carry out double-muscle phenotype sheep breeding. In order to solve the above-mentioned technical problems, the present invention firstly provides the application of the material for detecting polymorphisms or genotypes of SNP sites in the sheep genome in the production of products for identifying or assisting in the identification of sheep double-muscle phenotypic traits.

The SNP site is a SNP site in the sheep genome, which is the 235th nucleotide of sequence 1 in the sequence listing, which is A or G.

In order to solve the above-mentioned technical problem, the present invention also provides the application of the material for detecting the polymorphism or genotype of the SNP site in the sheep genome in identifying or assisting in identifying the phenotypic traits of double muscle in sheep.

The SNP site is a SNP site in the sheep genome, which is the 235th nucleotide of sequence 1 in the sequence listing, which is A or G.

In order to solve the above technical problems, the present invention also provides the application of the material for detecting the polymorphism or genotype of the SNP site in the sheep genome in sheep breeding or sheep breeding products.

The SNP site is a SNP site in the sheep genome, which is the 235th nucleotide of sequence 1 in the sequence listing, which is A or G.

Products containing the above-mentioned polymorphism or genotype detection material of the SNP molecular marker site in the sheep genome also belong to the protection scope of the present invention. The product can be any of the following G1)-G3):

G1) Products for detecting single nucleotide polymorphisms or genotypes related to double-muscle phenotypic traits in the sheep genome;

G2) Products for identifying or assisting in identifying the phenotypic traits of double muscle in sheep;

G3) Products for sheep breeding.

In order to solve the above technical problems, the present invention also provides a method for identifying or assisting in identifying that sheep have double-muscle traits. The method includes detecting the genotype of the above-mentioned SNP molecular marker site in the sheep genome to be tested, and identifying or assisting in identifying whether the sheep has a double-muscle trait according to the genotype of the SNP molecular marker site of the sheep to be tested: The sheep whose genotype of the genome SNP site of the test sheep is AA or GA are or candidates are sheep with double-muscle traits, and the genotype of the genome SNP site of the sheep to be tested is GG The sheep are or candidates do not have double-muscle traits. Muscular traits in sheep.

The application of the method described above in sheep breeding also belongs to the protection scope of the present invention.

The above-mentioned sheep can be at least one of Hu sheep, Kazakh sheep, Texel sheep, Dorper sheep, and Chinese Merino multi-parity fine-wool sheep.

The above-mentioned sheep breeding can be to breed sheep breeds with double-muscle traits.

The above-mentioned substances for detecting the polymorphism or genotype of the SNP molecular marker site can be the following D1), D2) or D3):

D1) PCR primers containing amplified sheep genomic DNA fragments including the SNP molecular marker site;

D2) PCR reagents containing the PCR primers described in D1);

D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2).

The PCR primers mentioned above can be a primer set consisting of the single-stranded DNA of sequence 2 in the sequence listing and sequence 3 in the sequence listing.

The kit described above may be a PCR kit. The PCR reagents of the kit may include: the above PCR primer set, 10×Taq buffer, Taq DNA polymerase, dNTP mix, sterilized deionized water (ddH ₂ O).

The embodiment of the present invention provides a molecular marker for identifying sheep double-muscle phenotype traits, which is located at the 235th position of sequence 1 in the sequence table of the sheep genome, where the nucleotide is A or G, and then the molecular marker is designed Relevant detection primers, according to the detection of the molecular marker in the sheep genome, determine the genotype of the sheep through the PCR product, which can effectively identify the double-muscle phenotype traits of the sheep: the genotype of the molecular marker in the genome is GA type (two bands ) or AA type sheep are candidate sheep with double-muscle phenotype traits, otherwise they are sheep without double-muscle phenotype traits.

Compared with prior art, the beneficial effect of the present invention is:

The molecular markers related to sheep double-muscle phenotype traits in the embodiment of the present invention are used for early breeding of sheep, and can be screened even when the sheep are just born, so as to improve the selection efficiency of sheep breeding with double-muscle phenotype traits and speed up the breeding process.

Description of drawings

Fig. 1 is the detection result of 2% agarose gel electrophoresis of the PCR amplification products of some sheep samples to be tested. Lanes 1 and 2 are the PCR amplification products of the Hu sheep genome; lanes 3 and 4 are the PCR amplification products of the Kazakh sheep genome; lanes 5 and 6 are the PCR amplification products of the Texel sheep genome; lanes 7 and 8 are the Dorper sheep Genomic PCR amplification products; Lanes 9, 10, and 11 are the genomic PCR amplification products of Chinese Merino multi-fetal meat fine-wool sheep.

Figure 2 is the SSCP electrophoresis profile of the PCR amplification product.

Figure 3 shows the sequencing results of different sheep PCR products.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The Hu sheep, Kazakh sheep, Texel sheep, Dorper sheep, and Chinese Merino fine-wool sheep for multi-fetal meat in the present invention can all be obtained from commercial sources.

Example 1. Molecular markers for identifying sheep double-muscle phenotypic traits and their applications

The SNP molecular marker is located in the MSTN gene of the sheep genome, and its flanking sequence is shown in sequence 1 in the sequence table, in sequence 1, r is a or g. The SNP molecular marker is located at the 235th position of sequence 1 in the sequence listing, where the nucleotide is A or G.

1. Sheep genome extraction and PCR amplification

A total of 336 blood samples were collected from 5 sheep breeds, including 48 Hu sheep, 48 Kazakh sheep, 56 Texel sheep, 57 Dorper sheep, and 127 Chinese Merino fine-wool sheep for multi-parity meat. Blood was collected from the jugular vein of each sheep, anticoagulated with ACD, and stored at -20°C. Genomic DNA of sheep was extracted using a genome extraction kit (Tiangen Biochemical Technology, Beijing, China, DP304) and tested for future use.

The following PCR system and PCR conditions were used for each sheep, and the genomic DNA of each sheep was used as a template to carry out PCR amplification to obtain PCR products. The PCR system is shown in Table 1 below. Among them, the primer pair used to detect the above molecular markers consists of two single-stranded DNAs (synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.), forward primer F and reverse primer R, and its sequence is as follows:

F: 5'-GTATTAAGGCACAAAGACAT-3' (sequence 2 in the sequence listing)

R: 5'-GAGTTAAATCATTTTGGTTTGC-3' (sequence 3 in the sequence listing)

Table 1 25μL PCR reaction system

In the PCR reaction system: 10×Taq buffer, Taq DNA polymerase, and dNTP mix were all provided by Sangon Bioengineering (Shanghai) Co., Ltd. (Cat. Nos. B600001 and B110047).

PCR reaction conditions: pre-denaturation at 94°C for 5 min; PCR cycle, denaturation at 94°C for 30 s, annealing at 49.5°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles, final extension at 72°C for 5 min, and storage at 4°C.

Get 5 μ L of the PCR amplification product of every sheep and carry out 2% agarose gel electrophoresis detection, gel imaging result shows: the PCR product of detected 336 sheep genomes all shows as 287bp in 2% agarose gel electrophoresis ( The specific band of sequence 1) in the sequence listing (Figure 1 is the result of agarose gel electrophoresis of PCR products of some samples).

2. Single Strand Conformation Polymorphism (SSCP) electrophoresis detection of PCR amplification products

2.1 SSCP electrophoresis detection method

Take 2 μL of the PCR product corresponding to each sheep obtained in 1.1, respectively, add 8 μL of denaturing buffer, denature at 98°C for 10 min, and quickly place it in ice for 10 min. Then the samples were added one by one into the sample wells of 10% non-denaturing polyacrylamide gel, and electrophoresed at 120 volts for 16 hours. After electrophoresis, turn off the electrophoresis apparatus and remove the gel for silver nitrate staining. The results of electrophoresis and silver staining are shown in Figure 2.

SSCP electrophoresis detection reagents include: 30% acrylamide, 10% ammonium persulfate, 5×TBE buffer, 0.5 MEDTA (pH8.0), denaturation buffer, fixative, staining solution, and developer. The preparation methods of the above-mentioned solutions are as follows, and all reagents are of domestic analytical grade.

30% acrylamide: weigh 290g of acrylamide, dissolve 10g of methylene bisacrylamide in 600mL of water, heat to dissolve (37°C), add distilled water to make up to 1000mL, store in a brown bottle at 4°C.

10% ammonium persulfate: Dissolve 10g of ammonium persulfate in 80mL of water, dilute to 100mL, store at 4°C.

5×TBE buffer solution: take 54g of Tris base, 2.75g of boric acid, 2mL of 0.5M EDTA (pH8.0) mother solution, and dilute to 1000mL with distilled water.

0.5M EDTA (pH8.0): Dissolve 186.1g disodium ethylenediaminetetraacetic acid dihydrate (EDTA-Na.2H2O) in 800mL water, stir vigorously on a magnetic stirrer, adjust the pH of the solution to 8.0 with NaOH, and then set Make up to 1L, autoclave, and store at 4°C.

Denaturation buffer: 0.01mol/L EDTA (pH8.0) 200μL, bromophenol blue 2.5mg, xylene cyanol 2.5mg, glycerin 200μL, 9.8mL deionized formamide and mix well.

Fixative solution: 2.5mL glacial acetic acid, 50mL absolute ethanol dissolved in 500mL water and mix well.

Staining solution: 1g silver nitrate, 2.5mL glacial acetic acid, 50mL absolute ethanol dissolved in 500mL water and mix well.

Developer solution: 15g NaOH, 2.5mL 37% formaldehyde dissolved in 500mL water and mix well.

2.2 SSCP electrophoresis detection band pattern analysis

The different sheep PCR products obtained in step 1 showed three different band types after SSCP electrophoresis and staining in step 2.1, and the genotypes corresponding to the different band types were defined as GG type, AA type and GA type. Among them, the GA type is displayed as two bands; the GG type is displayed as a band, and its position corresponds to the band located above the two bands of the GA type near the sampling hole; the AA type is displayed as a band, and its position corresponds to the two bands of the GA type. The lower part of the strip away from the well. Figure 2 shows the SSCP electrophoresis results of PCR products of some sheep with GG, GA and AA genotype individuals. In Fig. 2, the swimming lanes from left to right are the Hu sheep of GG genotype (the first swimming lane), the Texel sheep of AA genotype (the second swimming lane), and the Texel sheep of GA genotype (the first swimming lane). 3 lanes), GG genotype Kazakh sheep (4th lane), AA genotype Texel sheep (5th lane), GA genotype Texel sheep (6th lane).

Among the 48 Hu sheep detected, the PCR products of 48 showed one band in the SSCP gel, and its position corresponded to the band at the top of the two bands of the GA type, which was close to the sample well, and the band corresponded to the The genotype is GG type.

Among the 48 Kazakh sheep detected, the PCR products of 48 showed one band in the SSCP gel, and its position corresponded to the upper band of the two bands of the GA type, which was close to the spotting hole, and the band corresponded to the The genotype is GG type.

Among the 56 Texel sheep detected, the PCR products of 16 showed two bands in the SSCP gel, corresponding to the genotype GA; 40 of the PCR products showed in the SSCP gel It is a band whose position corresponds to the band at the bottom of the two bands of type GA and away from the spotting hole, and the genotype corresponding to this band is type AA.

Among the 57 Dorper sheep detected, the PCR products of 57 showed one band in the SSCP gel, and its position corresponded to the band at the top of the two bands of the GA type, which was close to the sample well, and the band corresponded to The genotype is GG type.

Among the 127 Chinese merino fine-wool sheep with multiple litters detected, the PCR products of 127 showed one band in the SSCP gel, and its position corresponded to the upper band of the two bands of the GA type near the sample well. Band, the genotype corresponding to this band is GG type.

The gene frequencies and genotype frequencies of different sheep breeds are shown in Table 2. Three band types (GG, AA, GA) appeared in the PCR products of the five sheep populations detected by SSCP electrophoresis. Among them, the GG genotype appeared only in Hu sheep, Kazak sheep, Dorper sheep, and Chinese Merino fine-wool sheep; there was no GG genotype in Texel sheep, and the frequency of A allele was significantly higher than that of G alleles.

Table 2. Gene frequency and genotype frequency distribution of different sheep breeds

The PCR products of each sheep were recovered and sequenced (Fig. 3), and the results showed that the genotypes of the SNP molecular marker sites of all AA type sheep individuals were AA (that is, the 235th SNP molecular marker of sequence 1 in the sheep genome sequence table The homozygous type whose locus is A); the genotype of all GA type sheep individuals is GA (that is, the heterozygous type whose SNP molecular marker loci at the 235th position of sequence 1 in the sheep genome sequence table is G and A); all GG The genotype of the individual sheep is GG (that is, the homozygous type whose SNP molecular marker site at the 235th position of sequence 1 in the sheep genome sequence table is G).

3. Determination of methods for SNP site identification or auxiliary identification of sheep with double-muscle phenotype traits

According to the SSCP band type of the PCR product, determine whether the detected sheep is a sheep with double-muscle phenotype traits according to the following method: If the SSCP band type of the PCR product to be identified shows GA type (two bands) or AA type (one band) Its position corresponds to the band of the two bands of GA type, which is located at the bottom and away from the spotting hole), and the sheep to be identified is a candidate sheep with double-muscle phenotype traits. If the SSCP band type of the PCR product of the sheep to be identified is displayed as GG type ( One band, its position corresponds to the upper band near the sampling hole in the two bands of GA type), then the sheep to be identified is a candidate sheep without double-muscle phenotype traits.

According to the above method, among the 48 Hu sheep detected, 48 GG type Hu sheep are all candidate sheep without double-muscle phenotype traits; among the 48 Kazak sheep detected, 48 GG type Kazak sheep are all candidates Sheep without double-muscle phenotype traits; among the 56 Texel sheep tested, 16 GA-type and 40 AA-type Texel sheep were candidate sheep with double-muscle phenotype traits; 57 Texel sheep tested Among the Dorper sheep, 57 Dorper sheep of GG type were all candidate sheep without double-muscle phenotype traits; among the 127 Chinese Merino multi-parity fine-wool sheep detected, 127 GG-type Chinese Merino multi-parity meat The fine-wool sheep used are all candidate sheep without double-muscle phenotype traits.

4. Verification of the accuracy of methods for identifying or assisting in identifying sheep with double-muscle phenotype traits

Observe the double-muscle phenotypes of 336 sheep of 5 sheep breeds collected in step 1 in the fattening stage.

The double-muscle phenotypes of 48 Hu sheep, 48 Kazakh sheep, 56 Texel sheep, 57 Dorper sheep, and 127 Chinese Merino fine-wool sheep were detected. The results show:

Among the 48 Hu sheep tested, 48 GG type Hu sheep had no obvious double muscle, and all of them belonged to sheep without double muscle phenotype traits.

Among the 48 Kazakh sheep tested, 48 Kazakh sheep of GG type had no obvious double muscle, and all belonged to sheep without double muscle phenotype traits.

Among the 56 Texel sheep tested, 16 GA type and 40 AA type Texel sheep had obvious double muscles, which belonged to the sheep with double muscle phenotype traits.

Among the 57 Dorper sheep tested, 57 Dorper sheep of GG type had no obvious double muscle, and all belonged to sheep without double muscle phenotype traits.

Among the 127 Chinese Merino multi-parity fine-wool sheep, 127 GG-type Chinese Merino multi-parity fine-wool sheep had no obvious double muscles, and all of them belonged to sheep without double-muscle phenotype traits.

Therefore the method that utilizes detection SNP molecular marker genotype detection sheep double-muscle phenotypic traits provided by the present invention is accurate and effective, can be used for the early stage breeding of sheep, even just can screen when sheep is just born, improves the double-muscle appearance character. The selection efficiency of sheep breeding for type traits can be improved, and the breeding process can be accelerated.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (10)

1. The application of the polymorphism or genotype of the SNP site in the sheep genome for the preparation of identification or auxiliary identification of sheep double-muscle phenotypic traits; the SNP site is a SNP site in the sheep genome, which is The 235th nucleotide of sequence 1 in the sequence listing is A or G. 2. The application of the polymorphism or genotype of the SNP site in the sheep genome to identify or assist in the identification of sheep double-muscle phenotypic traits; the SNP site is a SNP site in the sheep genome, which is a sequence listing The 235th nucleotide in sequence 1 is A or G. 3. The application of the polymorphism or genotype of the SNP site in the sheep genome to sheep breeding or sheep breeding products; the SNP site is a SNP site in the sheep genome, which is sequence 1 in the sequence table Nucleotide 235, which is A or G. 4. The product containing the polymorphism or the genotype of the detection sheep genome SNP molecular marker site described in claim 1 can be any one of the following G1)-G3): G1) Products for detecting single nucleotide polymorphisms or genotypes related to double-muscle phenotypic traits in the sheep genome; G2) Products for identifying or assisting in identifying the phenotypic traits of double muscle in sheep; G3) Products for sheep breeding. 5. A method for identification or auxiliary identification that sheep has double-muscle traits, comprising detecting the genotype of the SNP molecular marker site described in claim 1 in the sheep genome to be tested, according to the gene of the sheep SNP molecular marker site to be tested Type identification or auxiliary identification of whether the sheep has double-muscle traits: the genotype of the genome SNP site of the sheep to be tested is AA or GA. The sheep whose genotype is GG are or are candidates for the sheep without double-muscle trait. 6. Application of the method according to claim 5 in sheep breeding. 7. The application according to any one of claims 1-3 or 6, the product according to claim 4, and the method according to claim 5, characterized in that: the sheep are Hu sheep, Kazakh sheep, At least one of Texel sheep, Dorper sheep, and Chinese Merino multi-parity fine-wool sheep. 8. The application according to any one of claims 1-3 or 6, the product according to claim 4, and the method according to claim 5, characterized in that: said sheep breeding is to cultivate a double-muscle trait of sheep breeds. 9. The application according to any one of claims 1-3 or 6, the product according to claim 4, the method according to any one of claims 5 or 7, characterized in that: the detection of SNP molecular markers The polymorphism or genotype of the site is the following D1), D2) or D3): D1) PCR primers containing amplified sheep genomic DNA fragments including the SNP molecular marker site; D2) PCR reagents containing the PCR primers described in D1); D3) A kit containing the PCR primers described in D1) or the PCR reagents described in D2). 10. The application or product or method according to claim 9, characterized in that: the PCR primer is a primer set consisting of single-stranded DNA of sequence 2 in the sequence listing and sequence 3 in the sequence listing.

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