CN115820866A - Molecular marker, primer pair, kit and identification method related to sheep double muscle phenotype traits - Google Patents
Molecular marker, primer pair, kit and identification method related to sheep double muscle phenotype traits Download PDFInfo
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Abstract
The invention discloses a molecular marker, a primer pair, a kit and an identification method related to sheep double muscle phenotype traits. The invention provides a technical scheme for detecting polymorphism or genotype of SNP sites in a sheep genome and application of substances in preparation of products for identifying or assisting in identifying sheep double-muscle phenotypic characters; the SNP site is one SNP site of a sheep genome, is 235 th nucleotide of a sequence 1 in a sequence table, and is A or G. Experiments prove that the related detection primer of the molecular marker determines the genotype of the sheep through a PCR product according to the detection of the molecular marker in the sheep genome, can effectively identify the double-muscle phenotype character of the sheep, can be used for early breeding of the sheep, can be screened even when the sheep is born, improves the selection efficiency of breeding the sheep with the double-muscle phenotype character, and accelerates the breeding process.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker, a primer pair, a kit and an identification method related to sheep double muscle phenotype traits.
Background
The meat production performance of livestock is an important economic character in livestock production, and livestock workers continuously strive to breed varieties with high meat yield and good meat quality. Traditional breeding work is time-consuming and labor-consuming, but with the rapid development of life sciences and molecular biotechnology, researchers try to combine molecular breeding with traditional breeding methods to breed livestock varieties with obvious breakthrough in meat production performance.
Research shows that Myostatin (MSTN) can inhibit proliferation and differentiation of muscle cells, and has negative regulation and control effect on skeletal muscle development and regeneration. The MSTN gene is highly conserved in mammals, and overexpression reduces muscle mass; the skeletal muscle weight of MSTN-deficient livestock is increased, namely the so-called double-muscle phenotype, and double-muscle varieties which are bred for a long time due to MSTN gene mutation exist in the nature, such as Piezo and Belgian blue cattle. Currently, about 20 different types of genetic variations have been identified in mammalian MSTN genes, including deletions, insertions, and single nucleotide polymorphisms. The weight of skeletal muscle of MSTN-deficient and inhibited livestock is increased, the fat content is reduced, and the ratio of the muscle to other tissues is greatly improved.
The mutation of the MSTN gene causes the remarkable improvement of the meat production performance of livestock, so that the gene becomes a hotspot gene for research in the field of animal molecular breeding, and the targeted breeding of the double-muscle sheep with the MSTN mutation type has very important significance.
Disclosure of Invention
The technical problem to be solved by the invention is how to detect the double muscle phenotype characters of the sheep or how to carry out double muscle phenotype sheep breeding. In order to solve the technical problems, the invention firstly provides application of a substance for detecting polymorphism or genotype of SNP sites in a sheep genome in preparing products for identifying or assisting in identifying sheep double-muscle phenotypic characters.
The SNP site is one SNP site of a sheep genome, is 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
In order to solve the technical problems, the invention also provides application of the substance for detecting the polymorphism or genotype of the SNP site in the sheep genome in identification or auxiliary identification of sheep double muscle phenotype traits.
The SNP site is one SNP site of a sheep genome, is the 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
In order to solve the technical problems, the invention also provides application of the substance 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 one SNP site of a sheep genome, is the 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
Products containing the substances for detecting the polymorphism or genotype of the SNP molecular marker sites of the sheep genome also belong to the protection scope of the invention. The product may be any of the following G1) -G3):
g1 Products for detecting single nucleotide polymorphisms or genotypes associated with the double muscle phenotypic trait in the genome of sheep;
g2 Product for identifying or assisting in identifying a sheep double muscle phenotype trait;
g3 Products for sheep breeding.
In order to solve the technical problem, the invention also provides a method for identifying or assisting in identifying that the sheep has the double-muscle character. The method comprises the following steps of detecting the genotype of the SNP molecular marker locus in the genome of a sheep to be detected, and identifying or assisting in identifying whether the sheep has the double muscle character according to the genotype of the SNP molecular marker locus of the sheep to be detected: the sheep with the genotype of the genomic SNP locus of the sheep to be detected being AA or GA is or is selected as a sheep with the double muscle shape, and the sheep with the genotype of the genomic SNP locus of the sheep to be detected being GG is or is selected as a sheep without the double muscle shape.
The application of the method in sheep breeding also belongs to the protection scope of the invention.
The sheep may be at least one of a Hu sheep, a Kazak sheep, a Teckel sheep, a Dorpo sheep, and a fine wool sheep for merino polytomies in China.
The sheep breeding described above may be the breeding of sheep varieties with a double muscle trait.
The substance for detecting polymorphism or genotype at SNP molecular marker sites as described above may be D1), D2) or D3) as follows:
d1 Contain PCR primers for amplifying the sheep genomic DNA fragments including the SNP molecular marker sites;
d2 PCR reagents containing the PCR primers according to D1);
d3 A kit containing the PCR primers described in D1) or the PCR reagents described in D2).
The PCR primer can be a primer group consisting of single-stranded DNA of a sequence 2 in a sequence table and a sequence 3 in the sequence table.
The kit described above may be a PCR kit. The PCR reagents of the kit may include: the PCR primer set, 10 XTaq buffer solution, taq DNA polymerase, dNTP mix and sterilized deionized water (ddH) 2 O)。
The embodiment of the invention provides a molecular marker for identifying sheep double-muscle phenotype traits, which is positioned at 235 th position of a sequence 1 in a sheep genome, wherein the nucleotide is A or G, then a related detection primer of the molecular marker is designed, and the genotype of sheep is determined through a PCR product according to the detection of the molecular marker in the sheep genome, so that the sheep double-muscle phenotype traits can be effectively identified: sheep with the genotype of GA (two bands) or AA (AA) of the molecular marker in the genome are candidate sheep with the double muscle phenotype property, otherwise, sheep without the double muscle phenotype property.
Compared with the prior art, the invention has the beneficial effects that:
the molecular marker related to the double muscle phenotype character of the sheep is used for early breeding of the sheep, and can be screened even when the sheep are born, so that the selection efficiency of breeding the sheep with the double muscle phenotype character is improved, and the breeding process is accelerated.
Drawings
FIG. 1 shows the result of 2% agarose gel electrophoresis detection of PCR amplification products of a part of sheep samples to be detected. Lanes 1 and 2 are Hu sheep genome PCR amplification products; lanes 3 and 4 are the PCR amplification products of the Kazakh sheep genome; lanes 5 and 6 are PCR amplification products of the genome of the Teckessel sheep; lanes 7 and 8 are Dorper sheep genome PCR amplification products; lanes 9, 10, and 11 are PCR amplification products of the genome of fine-wool sheep used in merino foetal meat in China.
FIG. 2 is an SSCP electropherogram of the PCR amplification product.
FIG. 3 shows the sequencing results of the PCR products of different sheep.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The Hu sheep, the Kazak sheep, the texar sheep, the Dorper sheep and the Chinese merino polypoena mutton fine wool sheep can be obtained from commercial approaches.
Example 1 molecular marker for identifying sheep double-muscle phenotype character and application thereof
The SNP molecular marker is positioned in a sheep genome MSTN gene, the flanking sequence of the SNP molecular marker is shown as a sequence 1 in a sequence table, and in the sequence 1, r is a or g. The SNP molecular marker is positioned at position 235 of a sequence 1 in a sequence table, and the nucleotide is A or G.
1. Sheep genome extraction and PCR amplification
A total of 336 blood samples were collected from a total of 5 sheep species, including 48 hu, 48 kazak, 56 texel, 57 dupo, and 127 fine wool sheep for merino polytomies in china. Each sheep was collected via jugular vein, anticoagulated with ACD and stored at-20 deg.C. The sheep genome DNA was extracted using a genome extraction kit (Tiangen Biochemical technology, beijing, china, DP 304) and tested for future use.
And (3) carrying out PCR amplification on each sheep by respectively taking the genomic DNA of each sheep as a template by adopting the following PCR system and PCR conditions to obtain a PCR product. The PCR system is shown in Table 1 below. Wherein, the primer pair for detecting the molecular marker consists of two single-stranded DNAs (synthesized by Beijing Liuhua Dagenescience and technology Co., ltd.) of a forward primer F and a reverse primer R, and the sequences are as follows:
f5 'GTATTAAGGCACAAGACAT-3' (sequence 2 in sequence table)
R5 'GAGTTAAATCATTTTTTGGTTTGC-3' (SEQ ID NO: 3 in sequence table)
TABLE 1 25 μ L PCR reaction System
In a PCR reaction system: the 10 XTaq buffer, taq DNA polymerase and dNTP mix were supplied from Biotechnology engineering (Shanghai) Ltd. (products Nos. B600001 and B110047).
And (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 5min; PCR cycles, denaturation at 94 ℃ for 30s, annealing at 49.5 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles in total, final extension at 72 ℃ for 5min, and storage at 4 ℃.
Taking 5 mu L of PCR amplification product of each sheep to carry out 2% agarose gel electrophoresis detection, and the result of gel imaging shows that: the PCR products of 336 detected sheep genomes are all shown as a specific band of 287bp (sequence 1 in the sequence table) in 2% agarose gel electrophoresis (FIG. 1 is the agarose gel electrophoresis result of the PCR products of a part of samples).
Single-strand conformation polymorphism (SSCP) electrophoretic detection of PCR amplification product
2.1SSCP electrophoretic detection method
And respectively taking 2 mu L of PCR product corresponding to each sheep obtained in the step 1.1, respectively adding 8 mu L of denaturation buffer solution, quickly placing the mixture in ice bath for 10min after denaturation is carried out for 10min at the temperature of 98 ℃. Then, the samples were individually added to wells of 10% non-denatured polyacrylamide gel and electrophoresed at 120V for 16 hours. And (4) closing the electrophoresis apparatus after electrophoresis is finished, and taking down the gel for silver nitrate dyeing. The results of electrophoresis and silver staining are shown in FIG. 2.
The SSCP electrophoresis detection reagent comprises: 30% acrylamide, 10% ammonium persulfate, 5 XTBE buffer, 0.5MEDTA (pH8.0), denaturation buffer, stationary liquid, staining solution and developing solution. The above solutions were prepared as follows, wherein all reagents were domestic analytical grade.
30% acrylamide: 290g of acrylamide and 10g of methylene bisacrylamide are weighed and dissolved in 600mL of water, heated to dissolve (37 ℃) and supplemented with distilled water to reach the volume of 1000mL, and the mixture is placed in a brown bottle for storage at 4 ℃.
10% ammonium persulfate: 10g of ammonium persulfate is dissolved in 80mL of water, the volume is determined to be 100mL, and the solution is stored at 4 ℃.
5 × TBE buffer: 54g of Tris base, 2.75g of boric acid and 2mL of 0.5M EDTA (pH8.0) mother liquor are taken, and the volume is adjusted to 1000mL by using distilled water.
0.5M EDTA (pH8.0): dissolving 186.1g disodium ethylene diamine tetraacetate dihydrate (EDTA-Na.2H2O) in 800mL water, vigorously stirring on a magnetic stirrer, adjusting the pH of the solution to 8.0 by using NaOH, then fixing the volume to 1L, autoclaving, and storing at 4 ℃.
Denaturation buffer: 0.01mol/L EDTA (pH8.0) 200. Mu.L, bromophenol blue 2.5mg, xylene blue 2.5mg, glycerol 200. Mu.L, 9.8mL deionized formamide.
Fixing liquid: 2.5mL of glacial acetic acid and 50mL of absolute ethyl alcohol are dissolved in 500mL of water and mixed uniformly.
Dyeing liquor: 1g of silver nitrate, 2.5mL of glacial acetic acid and 50mL of absolute ethyl alcohol are dissolved in 500mL of water and mixed uniformly.
Developing solution: 15gNaOH,2.5mL 37% formaldehyde dissolved in 500mL water mixing.
2.2SSCP electrophoretic detection of banding patterns
And (3) allowing different sheep PCR products obtained in the step (1) to respectively generate 3 different banding patterns after SSCP electrophoresis and dyeing in the step (2.1), and defining genotypes corresponding to the different banding patterns as GG type, AA type and GA type. Wherein GA type is shown as two bands; GG type shows a band corresponding to the upper band of the two GA type bands near the spot wells; type AA shows a band corresponding in position to the lower, well-spaced band of the two GA-type bands. FIG. 2 shows SSCP electrophoresis results of PCR products of a portion of sheep individuals with GG, GA, and AA genotypes. In FIG. 2, from left to right, the lane is Hu sheep with GG genotype (lane 1), techholr sheep with AA genotype (lane 2), techholr sheep with GA genotype (lane 3), kazakh sheep with GG genotype (lane 4), techholr sheep with AA genotype (lane 5), and Techholr sheep with GA genotype (lane 6).
Of the 48 Hu sheep tested, 48 PCR products were shown on SSCP gels as one band, which corresponded to the upper band of the two GA-type bands near the well, and the genotype of this band was GG-type.
Of the 48 examined Kazakh sheep, 48 PCR products showed a single band on the SSCP gel at a position corresponding to the upper band of the two GA-type bands near the wells, and the genotype of the band was GG-type.
Of the 56 detected texels, 16 PCR products were shown as two bands in the SSCP gel, with the corresponding genotype GA; 40 PCR products showed a band on the SSCP gel corresponding to the lower of the two GA-type bands far from the well, and the genotype of this band was AA-type.
Of the 57 Dorper sheep tested, 57 PCR products were shown on SSCP gels as one band corresponding to the upper band of the two GA-type bands near the wells, which corresponds to the GG-type genotype.
In the 127 tested merino babyss of china, 127 PCR products were shown to be one band on the SSCP gel, corresponding to the upper band near the spot well of the two GA-type bands, and the genotype of the band was GG-type.
The gene frequencies and genotype frequencies for different sheep breeds are shown in table 2.PCR products of 5 sheep groups detected showed 3 banding patterns (GG, AA, GA) by SSCP electrophoresis. Wherein the Hu sheep, the Kazak sheep, the Dorper sheep and the Chinese merino mullet mutton fine wool sheep only have GG genotype; there was no occurrence of the GG genotype in the Teckessel sheep, and the frequency of occurrence of the A allele was significantly higher than that of the G allele.
TABLE 2 Gene frequencies and genotype frequency distributions for different sheep breeds
The PCR products were recovered separately from each sheep and sequenced (fig. 3), indicating that: the genotypes of the SNP molecular marker loci of all AA type sheep individuals are AA (namely the 235 th SNP molecular marker locus of the sequence 1 in the sheep genome sequence table is homozygous for A); the genotypes of all GA type sheep individuals are GA (namely the 235 th SNP molecular marker site of the sequence 1 in the sheep genome sequence table is a heterozygote of G and A); the genotypes of all GG sheep individuals are GG (i.e. the homozygous type with the 235 th SNP molecular marker site of the sequence 1 in the sheep genome sequence table as G).
Determination of method for identifying SNP locus or identifying sheep with double muscle phenotype character in auxiliary mode
And (3) determining whether the detected sheep is the sheep with the double muscle phenotype character according to the SSCP banding pattern of the PCR product according to the following method: if the SSCP banding pattern of the PCR product of the sheep to be identified is shown as GA type (two bands) or AA type (one band, the position of which corresponds to the lower band far away from the spotted hole in the two GA type bands), the sheep to be identified is a sheep candidate with the double muscle phenotype character, and if the SSCP banding pattern of the PCR product of the sheep to be identified is shown as GG type (one band, the position of which corresponds to the upper band near the spotted hole in the two GA type bands), the sheep to be identified is a sheep candidate without the double muscle phenotype character.
Determining that 48 detected Hu sheep are candidate sheep without the double muscle phenotype character from 48 detected Hu sheep; of the 48 detected Kazakh sheep, 48 GG type Kazakh sheep were all candidate sheep without the double-muscle phenotype character; 16 GA-type and 40 AA-type of 56 detected Teckel sheep are candidate sheep with the double muscle phenotype character; among 57 detected doherty sheep, 57 GG-type doherty sheep are all candidate sheep without the double-muscle phenotype character; of the 127 tested chinese merino multiparous mutton fine hair sheep, 127 GG type chinese merino multiparous mutton fine hair sheep were all candidate sheep without the double muscle phenotype trait.
4. Accuracy verification of method for identifying or assisting in identifying sheep with double-muscle phenotypic characters
And (3) observing the double muscle phenotype of the total 336 sheep fattening stages of the 5 sheep varieties collected in the step 1.
The double muscle phenotype of 48 Hu sheep, 48 Kazak sheep, 56 Texel sheep, 57 DuPo sheep, and 127 Chinese merino multiparous mutton fine hair sheep was examined. The results show that:
of the 48 detected Hu sheep, 48 GG type Hu sheep have no obvious double muscle and belong to sheep without the double muscle phenotype character.
Of the 48 tested Kazakh sheep, 48 GG type Kazakh sheep had no obvious double muscle and all were sheep without double muscle phenotype character.
Of the 56 examined texels, 16 GA-type and 40 AA-type texels were significantly sarcoid and belonged to sheep with a sarcoid phenotypic trait.
Of the 57 Dorper sheep tested, 57 GG-type Dorper sheep had no apparent double muscle and were all sheep with no double muscle phenotype.
Among 127 detected chinese merino multiparous meat fine-hair sheep, 127 GG type chinese merino multiparous meat fine-hair sheep had no obvious double muscles, and all of them were sheep without double muscle phenotype character.
Therefore, the method for detecting the sheep double-muscle phenotype character by using the SNP molecular marker genotype is accurate and effective, can be used for the early breeding of sheep, can be used for screening even at the birth of sheep, improves the selection efficiency of sheep breeding with the double-muscle phenotype character, and accelerates the breeding process.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The application of the substance for detecting the polymorphism or genotype of the SNP site in the sheep genome in preparing products for identifying or assisting in identifying the sheep double-muscle phenotype property; the SNP site is one SNP site of a sheep genome, is 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
2. The application of the substance for detecting the polymorphism or genotype of the SNP locus in the sheep genome in identifying or assisting in identifying the sheep double muscle phenotype traits; the SNP site is one SNP site of a sheep genome, is 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
3. The application of the substance for detecting the polymorphism or genotype of the SNP locus in the sheep genome in sheep breeding or sheep breeding products; the SNP site is one SNP site of a sheep genome, is 235 th nucleotide of a sequence 1 in a sequence table, and is A or G.
4. The product containing the substance for detecting the polymorphism or the genotype of the SNP molecular marker site of the sheep genome according to claim 1 can be any one of the following G1) to G3):
g1 Products for detecting single nucleotide polymorphisms or genotypes associated with the double muscle phenotypic trait in the genome of sheep;
g2 Product for identifying or assisting in identifying a sheep double muscle phenotype trait;
g3 Products for sheep breeding.
5. A method for identifying or assisting in identifying that sheep has a double-muscle trait, which comprises detecting the genotype of the SNP molecular marker site in claim 1 in the genome of a test sheep, and identifying or assisting in identifying whether the sheep has the double-muscle trait according to the genotype of the SNP molecular marker site of the test sheep: the sheep with the genotype of the genomic SNP locus of the sheep to be detected being AA or GA is or is selected as a sheep with the double muscle shape, and the sheep with the genotype of the genomic SNP locus of the sheep to be detected being GG is or is selected as a sheep without the double muscle shape.
6. Use of the method of claim 5 in sheep breeding.
7. Use according to any of claims 1-3 or 6, product according to claim 4, method according to claim 5, characterized in that: the sheep is at least one of Hu sheep, kazak sheep, texel sheep, dorpo sheep and Chinese merino polyplasia sheep.
8. Use according to any of claims 1-3 or 6, product according to claim 4, method according to claim 5, characterized in that: the sheep breeding is to breed sheep varieties with double-muscle characters.
9. The use of any one of claims 1 to 3 or 6, the product of claim 4, the method of any one of claims 5 or 7, wherein: the substances for detecting the polymorphism or genotype of the SNP molecular marker sites are D1), D2) or D3) as follows:
d1 Contain PCR primers for amplifying the sheep genomic DNA fragments including the SNP molecular marker sites;
d2 PCR reagents containing the PCR primers according to D1);
d3 A kit containing the PCR primers described in D1) or the PCR reagents described in D2).
10. The use or product or method according to claim 9, wherein: the PCR primer is a primer group consisting of single-stranded DNA of a sequence 2 in a sequence table and a sequence 3 in the sequence table.
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