CN117385060B - SNP marker related to sheep growth performance and application thereof - Google Patents
SNP marker related to sheep growth performance and application thereof Download PDFInfo
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- 238000010586 diagram Methods 0.000 description 6
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- 108090000623 proteins and genes Proteins 0.000 description 5
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- 102100037852 Insulin-like growth factor I Human genes 0.000 description 2
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- 102220043531 rs74637005 Human genes 0.000 description 2
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Abstract
The invention relates to the technical field of animal breeding, in particular to an SNP marker related to sheep growth performance and application thereof. The SNP marker is positioned at the 324 th position of a nucleic acid, and the nucleic acid has a nucleotide sequence shown as SEQ ID NO. 1. The SNP marker provided by the invention is closely related to the growth performance of sheep, and the identification of the growth performance of sheep can be realized through the detection of the SNP marker. The SNP marker provided by the invention can be applied to molecular marker assisted breeding of sheep, and has important significance to the sheep breeding field.
Description
Technical Field
The invention relates to the technical field of animal breeding, in particular to an SNP marker related to sheep growth performance and application thereof.
Background
A single nucleotide polymorphism (single nucleotide polymorphism, SNP) refers to a polymorphism in a DNA sequence at the genomic level caused by a single base variation that can be inherited to offspring. Variations caused by a portion of the individual nucleotides may result in changes in gene function and thus in phenotypic changes in the individual.
SOCS2 (suppressor of cytokine signaling) is a functional gene related to animal growth rate and size, and the gene affects the growth traits of individuals by negatively regulating GH/IGF1 signal axes, so when the function of the SOCS2 gene is affected, the negative regulation signal to the GH/IGF1 signal axes is weakened, and finally the growth rate of individuals is accelerated, the size is increased, the 96 th amino acid p96.R > C mutation of SOCS2 is reported in French sheep, the weight and size of individuals can be increased, but no mutation at the position is reported in sheep breeds in China.
The growth character is one of the most important indexes for breeding the meat sheep variety, and the breeding of the sheep strain with high growth speed and large body size is beneficial to improving the production efficiency and the industrialization level. If the molecular marker is applied to mutton sheep breeding, individuals with better growth traits are required to be identified for breeding by utilizing the molecular marker, but the molecular marker and the corresponding detection method which are suitable for breeding the growth speed and the size of domestic local sheep varieties are lacking at present because SOCS2 p.R96C point mutation is not found in the Chinese sheep varieties.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an SNP marker related to sheep growth performance and application thereof.
In a first aspect, the present invention provides a SNP marker of base 129558029 of sheep chromosome 3, wherein the polymorphism is A/C.
Further, the reference genome version number is: far_v4.0 (GCF 000298735.2) sequence number NC019460.2.
Further, the SNP marker is located at position 324 of a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO. 1.
Further, the sheep having the SNP marker polymorphism A has a higher growth performance than the sheep having the SNP marker polymorphism C.
In a second aspect, the present invention provides a primer pair comprising:
the upstream primer SOE2F1:5'-cattgttgccaagtacttgccct-3' the number of the individual pieces of the plastic,
downstream primer SOE2R1:5'-gttccttcagcctttacttgggatg-3'.
The invention further provides a kit comprising the SNP marker, or the primer pair.
In a third aspect, the invention provides the use of said SNP marker, or said primer pair, or said kit, in the detection of sheep growth performance.
The invention further provides the SNP marker, or the primer pair, or the application of the kit in sheep breeding.
In a fourth aspect, the invention provides a method of detecting growth performance of sheep comprising:
and detecting the SNP marker of the sheep to be detected, and judging the growth performance of the sheep to be detected according to a detection result.
Further, the method comprises the steps of:
extracting genome DNA of the sheep to be detected; carrying out PCR amplification by using the primer pair, and detecting the genotype of the SNP marker of the sheep to be detected; judging the growth performance of the sheep to be tested according to the genotype detection result.
Further, the judging the growth performance of the sheep to be tested according to the genotype detection result comprises:
sheep with genotype test AC showed better growth performance than sheep with genotype test CC.
Further, the detecting the genotype of the SNP marker of the sheep to be tested includes:
sequencing the PCR amplification product.
Further, the growth performance includes:
one or more of body weight, body diagonal length, chest circumference or width.
The invention has the following beneficial effects:
the existing SOCS2 mutation site R96C related to sheep growth traits is reported in French sheep and is not present in a plurality of Chinese local sheep varieties. The invention researches and obtains a new SNP marker locus related to sheep growth performance, and can realize the identification of sheep growth performance by detecting the marker locus, fills the blank of domestic sheep growth-free character related molecular markers, and has important value for breeding sheep strains with higher growth performance.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a Manhattan diagram of whole genome resequencing provided in example 1 of the present invention.
FIG. 2 is a schematic diagram of SOCS2 gene and primer design according to example 2 of the present invention.
FIG. 3 is a schematic diagram showing the detection results of PCR products of wild-type and mutant sheep genomes provided in example 2 of the present invention; wherein the wild type is sheep without the g.129558029 point mutation molecular marker, and the mutant type is sheep with the g.129558029C > A point mutation molecular marker.
FIG. 4 is a schematic representation of the sequencing results of the wild-type and mutant sheep genomic PCR products provided in example 2 of the present invention; wherein g.129558029C is sheep marked by a molecule without point mutation; g.129558029C > A is sheep marked with a point mutation molecule.
FIG. 5 is a schematic diagram showing the birth weight and the weight of different months of age of a heterozygous individual containing the g.129558029C > A point mutation molecular marker provided in example 2 of the present invention; wherein PM is sheep containing g.129558029C > A point mutation molecular markers; WT is a wild-type sheep that does not contain a g.129558029c > a point mutation molecular marker.
FIG. 6 is a schematic diagram showing the comparison of a heterozygous individual containing a g.129558029C > A point mutation molecular marker provided in example 3 of the present invention with a wild-type individual not containing a g.129558029C > A point mutation molecular marker for height, length of body inclination, chest circumference and width of the individual; wherein SOCS2 PM is sheep containing a g.129558029C > A point mutation molecular marker, and WT is a wild-type sheep not containing a g.129558029C > A point mutation molecular marker.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. Obtaining SNP molecular marker locus g.129558029C > A
The invention performs Genome-wide re-sequencing and Genome-wide selective sweep by collecting blood genomes of 30 Hu sheep, 20 Hu-lun Bei Eryang, 20 Su-nit sheep and 10 small-tailed han sheep (all Chinese local sheep variety), and the result shows that a segment near 3 exon SOCS2 in Hu-lun-Baker sheep has continuous selection signals (figure 1), namely, some gene mutations in the segment are naturally or manually selected in the domestication or feeding process of Hu-lun Bei Eryang, and further analysis finds that the segment contains a mutation site (g.129558029C > A) in a key domain SH2 (Scrhology 2 phosphotyrosine binding domain) of SOCS2, and the amino acid mutation in the region possibly leads to SOCS2 functional inactivation, thereby influencing sheep growth, so that whether an individual with the mutation has higher growth performance is verified in individuals when the selected site.
Specifically, the invention provides a SNP molecular marker locus g.129558029C > A (polymorphism is A/C, located at 129558029 base of sheep chromosome 3). The reference genome version numbers are: far v4.0 (GCF 000298735.2) sequence number NC019460.2.
2. Mutation frequency of SNP molecular marker site g.129558029C > A in large group
The invention collects blood in 200 Hu-sheep Bei Eryang and 200 Hu-sheep, carries out genome extraction, PCR amplification and sanger sequencing (the method is consistent with that in the patent), identifies genotypes of all sheep, counts genotype frequencies and gene frequencies, and proves that the mutation frequency of the SNP locus in sheep is lower, namely 0.02 in Hu-sheep and 0.01 in Hu-Lun-bell sheep respectively, and has no obvious difference (P > 0.05) in two sheep varieties (see table 1).
TABLE 1 genotype frequencies of SNP molecular marker loci g.129558029C > A in large populations
Example 2
The relation between SNP molecular marker locus g.129558029C > A and sheep growth performance is verified, and a SOCS2 gene and primer design schematic diagram is shown in figure 2, and specifically comprises the following procedures:
(1) Detection flow
The primers used were:
the upstream primer SOE2F1:5'-cattgttgccaagtacttgccct-3' the number of the individual pieces of the plastic,
downstream primer SOE2R1:5'-gttccttcagcctttacttgggatg-3'.
The amplification system is as follows:
amplification was performed using high-fidelity PCR enzyme (2 XPhanta Max Master Mix) from Nanjinouzan Biotech, inc., cat# P525-02.
TABLE 2 SOCS2 exon2 amplification System
The amplification conditions were: amplification was performed using a PCR amplification instrument, first stage: pre-denaturation at 95 ℃ for 5min; the second stage of denaturation at 95℃for 30s, annealing at 58℃for 30s and elongation at 72℃for 45s,34 cycles; the third stage was carried out at 72℃for 5 minutes and stored at 4 ℃.
FIG. 3 is an example of primer amplification results, wherein the size of a wild sheep genome PCR product without the g.129558029C > A point mutation molecular marker is 824bp, and the size of a mutant sheep genome PCR product with the g.129558029C > A point mutation molecular marker is 824bp.
Further, the PCR amplified product obtained by the primers is subjected to sanger sequencing by using an F1 primer, and is compared with a reference gene sequence, and a comparison example result is shown in FIG. 4, wherein the genotype of the wild sheep without the g.129558029C > A point mutation molecular marker at the locus is C; the genotype of the mutant sheep with g.129558029C > A point mutant molecular marker at the site is A.
(2) Result verification
The invention is based on the method to amplify genome of 16 sheep (the breed is Hu sheep), 7 heterozygous individuals containing g.129558029C > A point mutation molecular markers are obtained after sequencing PCR products, and 9 wild individuals do not contain g.129558029C > A point mutation molecular markers.
The invention further detects the birth weight of 7 heterozygous individuals containing g.129558029C > A point mutation molecular markers and obtains the average value; the birth weight and the weight of different ages of the day were measured for 9 wild-type individuals having the same sex and the same age of the day, and an average value was obtained. The growth curve was plotted to give the results shown in FIG. 5, and from the results, heterozygous individuals containing the g.129558029C > A point mutation molecular marker had significantly higher birth weights and different month-old weights than wild-type individuals.
Example 3
In this example, 20 sheep (race: hulan Bei Eryang) genomes were subjected to detection of g.129558029C > A point mutation molecular markers based on the method provided in example 1, and after sequencing of PCR products, 4 sheep were obtained as heterozygous individuals containing g.129558029C > A point mutation molecular markers, and 16 sheep with the same gender and the same age of day were wild type individuals (not containing g.129558029C > A point mutation molecular markers). For heterozygous individuals and wild individuals containing the g.129558029C > A point mutation molecular marker, detecting the height, the body diagonal length, the chest circumference and the width of the individuals, taking an average value and plotting.
The results are shown in fig. 6, and from the results, heterozygous individuals comprising the g.129558029C > a point mutation molecular marker were significantly higher in body diagonal length, chest circumference and jirimwidth than wild type individuals.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A SNP marker is characterized in that the nucleotide sequence of the SNP marker is shown as SEQ ID NO.1, wherein the 324 th polymorphism is A/C.
2. The SNP marker according to claim 1, wherein the SNP marker is base 129558029 of sheep chromosome 3 and the polymorphism is A/C.
3. The SNP marker according to claim 1 or 2, wherein the sheep having SNP marker polymorphism a has higher growth performance than the sheep having SNP marker polymorphism C.
4. A kit comprising the SNP marker of any one of claims 1-3.
5. Use of the SNP marker of any one of claims 1-3 or the kit of claim 4 for the detection of sheep growth performance.
6. Use of the SNP marker of any one of claims 1-3 or the kit of claim 4 in sheep breeding.
7. A method of detecting sheep growth performance comprising:
judging the growth performance of the sheep to be tested according to the detection result by detecting the SNP marker according to any one of claims 1 to 3.
8. The method according to claim 7, comprising:
extracting genome DNA of the sheep to be detected; performing PCR amplification by using a primer pair, and detecting the genotype of the SNP marker according to any one of claims 1 to 3 of the sheep to be detected; judging the growth performance of the sheep to be tested according to genotype detection results;
the primer pair comprises:
the upstream primer SOE2F1:5'-cattgttgccaagtacttgccct-3' the number of the individual pieces of the plastic,
downstream primer SOE2R1:5'-gttccttcagcctttacttgggatg-3'.
9. The method of claim 8, wherein determining the growth performance of the sheep under test based on the genotype test results comprises:
sheep with genotype test AC showed better growth performance than sheep with genotype test CC.
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