CN115011676A - Detection method and application of genetic polymorphic site related to sheep meat tenderness - Google Patents

Detection method and application of genetic polymorphic site related to sheep meat tenderness Download PDF

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CN115011676A
CN115011676A CN202210654294.4A CN202210654294A CN115011676A CN 115011676 A CN115011676 A CN 115011676A CN 202210654294 A CN202210654294 A CN 202210654294A CN 115011676 A CN115011676 A CN 115011676A
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肖成
刘宇
曹阳
柳俭强
苑志宇
张立春
马惠海
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Jilin Academy of Agricultural Sciences
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Abstract

The invention discloses a detection method and application of a genetic polymorphic site related to the tenderness of sheep meat. Through the analysis of genetic polymorphism of the sheep CCN5 gene in a population by a sequencing technology and the combination of individual meat quality character data for correlation analysis, the sheep population with dominant meat tenderness can be early selected according to polymorphic sites on the 5 th exon of the CCN5 gene.

Description

Detection method and application of genetic polymorphic site related to sheep meat tenderness
Technical Field
The invention belongs to the field of gene detection and livestock and poultry variety improvement and breeding, and particularly relates to identification of a Single Nucleotide Polymorphism (SNP) marker related to tenderness of sheep meat.
Background
With the economic progress, the living standard of residents is continuously improved, the catering industry is developed vigorously, and the mutton is more popular with the public. The requirements of people on meat quality are increased while the demand of mutton is increased. In recent years, breeders have gradually shifted the direction of research to the improvement of sheep meat quality. The overall quality of mutton is comprehensively influenced by various factors, for example, the tenderness of the mutton can be obviously influenced by the intramuscular fat content; unsaturated fatty acid in mutton can not only influence the flavor of mutton, but also be necessary nutrient substances for human bodies. By ascertaining the candidate genes influencing the sheep meat quality character and the relevance of the mutation sites on the candidate genes and the sheep colony meat quality character difference, an important basis is provided for molecular breeding work of improving the sheep meat quality. Early studies revealed the expression rule of genes related to the differentiation process of preadipocytes of small tailed han sheep (DOI:10.16431/j. cnki.1671-7236.2019.07.018.), but major genes affecting meat quality traits have not yet been determined.
Intercellular communication network factor 5 (CCN 5) is involved in various physiological processes of the body, such as cell adhesion, proliferation, differentiation, migration, maturation, etc. Research shows that the CCN5 gene can influence the proliferation and differentiation of Mesenchymal Stem Cells (MSCs) and participate in fat metabolism of organisms. However, the CCN5 gene has been studied less on livestock meat quality, and the regulation and control effect on the meat quality traits of the body is still unclear. Currently, the search for the SNP marker of the CCN5 gene is in a state of being arrested.
Disclosure of Invention
The invention aims to provide a detection method and application of genetic polymorphic sites related to the meat tenderness of sheep, provides a quick and effective method for selecting sheep individuals with relatively high intramuscular fat content in a fixed population, and provides a basis for breeding for improving the meat quality of sheep.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting single nucleotide polymorphism of sheep CCN5 gene comprises the following steps:
a partial fragment of a CCN5 gene is amplified by PCR by taking sheep genome DNA or DNA contained in sheep in-vitro biological materials as a template, and the genotype of the 5 th exon single nucleotide polymorphism site of the CCN5 gene is identified by using the fragment.
Preferably, the single nucleotide polymorphism site is located at chr.13:7625253, which site has a C-G mutation in a sheep (e.g., small tailed Han sheep) population.
Preferably, the primers for PCR are:
an upstream primer: 5'-AACGGAGGGAAGGACATCAAAT-3' (namely SEQ. ID. NO.1)
A downstream primer: 5'-AGCGGCTGACATAGAGTTTCCA-3' (i.e. SEQ. ID. NO. 2).
Preferably, the sheep excised biological material is blood collected from a sheep individual.
Preferably, the reaction system of the PCR comprises: 50-150 ng/. mu.L sheep genome DNA 1-2. mu.L or sheep blood sample 1-2. mu.L, and 10-20. mu. mol/L upstream and downstream primers 0.4-0.5. mu.L respectively.
Preferably, the reaction conditions of the PCR include: 5min at 94 ℃; 35 cycles of 94-95 ℃ for 30s, 55-65 ℃ for 30s and 72 ℃ for 16 s; and 8min at 72 ℃.
Preferably, the identification of the genotype comprises the following steps: the fragments are sequenced and the sequencing result is then aligned with the reference sequence.
A kit for detecting a single nucleotide polymorphism of a sheep CCN5 gene, which comprises site amplification primers (e.g., the above-mentioned upstream primer and downstream primer) for identifying the genotype of the 5 th exon single nucleotide polymorphism site of the CCN5 gene by a PCR-sequencing method.
The detection method of the single nucleotide polymorphism of the sheep CCN5 gene is applied to sheep molecular marker-assisted selective breeding.
Preferably, the individual with the single nucleotide polymorphism site (specifically chr.13:7625253) with the genotype of CG or CC is superior in meat quality traits.
Preferably, the meat quality trait is selected from the group consisting of intramuscular fat content.
Preferably, the sheep are selected from small tailed han sheep.
The invention has the beneficial effects that:
according to the SNP locus located in the 5 th exon of the CCN5 gene, the rapid and accurate typing of the CCN5 gene of a sheep (such as small tailed han sheep) population can be realized. And based on the obvious correlation between the site and the individual meat quality traits, the molecular marker (namely SNP marker) can be used for screening sheep individuals with good meat quality (such as tenderness) at an early stage, so that the breeding speed of the dominant mutton sheep (such as small tailed Han sheep) is increased.
Furthermore, compared with the traditional typing method, the method for identifying the genotype by sequencing has the advantages of short time consumption, high success rate, more visual result and capability of carrying out identification and selection in a large scale.
Drawings
FIG. 1 is an electrophoretogram of amplified products of the 5 th exon region of the CCN5 gene of small tailed han sheep; the 5 lanes to the right of Marker in the figure are different individual samples.
FIG. 2 is a sequence chart of a region of exon5 of the CCN5 gene of small tailed han sheep; in the figure, WE5-25-W5R _ E02/F02/G02/H02/A03/B03/C03.ab1RC represents different individual samples.
FIG. 3 is a sequencing peak diagram of different genotypes of the CCN5 gene (exon 5) of small tailed han sheep; wherein: a is CC genotype, B is GG genotype, and C is CG genotype.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
(I) sheep genome candidate region screening
In the invention, a representative small tailed Han sheep of mutton sheep variety is selected as an experimental object. Detecting the differential expression genes in the differentiation process of the experimental sheep adipocytes by a transcriptome sequencing technology in the early stage of a laboratory. The result shows that the CCN5 gene is related to fat metabolism of experimental sheep and can influence the meat quality traits of the experimental sheep, and then the later discovery shows that no related report exists before. Subsequent experimental tests show that only the 5 th exon of the CCN5 gene has one potential SNP site. Therefore, the site is analyzed by being associated with experimental mutton data, and an attempt is made to prove that the CCN5 gene has an SNP marker related to the sheep meat quality trait (see the experimental contents below in detail).
(II) sheep sample collection and detection of exon5 mutation of CCN5 gene
1. Sample collection
Blood samples were collected from small tailed han sheep raised by the college of agriculture academy of Jilin province, Changchun, 2021 month 10. Blood samples are obtained by collecting blood through jugular veins, and the collected blood is stored in an anticoagulation tube and stored in a refrigerator at 4 ℃; 96 small-tail Han sheep of 6 months age were collected.
2. Mutation detection strategy
Designing a specific primer according to a sheep CCN5 gene sequence, cloning a DNA fragment of the 5 th exon region of a CCN5 gene by PCR, detecting the DNA fragment by using a direct sequencing technology to obtain sequence information, and judging the 5 th exon mutation condition of the CCN5 gene of a sample according to a sheep genome reference sequence.
3. Primer design
1 pair of primers are designed by taking the DNA sequence of the sheep CCN5 gene as a reference (the gene ID number is XM-027977228.2), the sequences of the primers are as follows, and the length of a target fragment is 745 bp:
CCN5 gene exon5 upstream primer: 5'-AACGGAGGGAAGGACATCAAAT-3'
Downstream primer of exon5 of CCN5 gene: 5'-AGCGGCTGACATAGAGTTTCCA-3'
Primers were synthesized by Jinzhi biology, Inc., Suzhou.
PCR amplification
Extracting genome DNA from collected small tailed han sheep blood samples, and determining the optimal test temperature by using a gradient PCR test by using the genome DNA as a template.
Add PCR reaction system to 200. mu.L of PCR centrifuge tube: 2 × Master Mix 10 μ L, forward primer 0.5 μ L (10 μmol/L), reverse primer 0.5 μ L (10 μmol/L), genomic DNA 1 μ L (about 50ng/μ L), in ddH 2 The content of O is filled to 20 mu L.
Placing a PCR centrifugal tube in a PCR instrument, and setting reaction conditions as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; gradient annealing is carried out for 30s at the temperature of 55-65 ℃; extension at 72 ℃ for 16s, 35 cycles starting from the second step; after circulation is finished, extension is carried out for 8min at 72 ℃; storing at 4 ℃.
mu.L of the PCR product was electrophoresed on 2% agarose gel for 20min at 1 XTBE. And determining the final annealing temperature according to the brightness and the definition of the strip.
After the final annealing temperature is determined, a full-scale gold blood PCR kit is utilized, collected sheep blood samples are taken as templates, and target fragment amplification is carried out according to the following PCR reaction system:
2 × Master Mix 10 μ L, 10 μmol/L upstream and downstream primers 0.4 μ L each, blood volume 1 μ L, and use ddH 2 The content of O is supplemented to 20 mu L.
The preparation of the PCR reaction system is carried out on ice, and the mixture is put into a PCR instrument after being fully and evenly mixed. The final PCR reaction conditions were: 5min at 94 ℃; annealing at 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 16s for 35 cycles; and 8min at 72 ℃.
After the reaction, 5. mu.L of the PCR product was detected by electrophoresis on 1 XTBE using 2% agarose gel, and the detection results are shown in FIG. 1.
5. Fragment sequencing and sequence analysis
The 745bp target fragment amplified by PCR was band-recovered and directly subjected to Sanger sequencing.
Referring to FIG. 2, the sequencing result of the sample is compared with the reference genome sequence (gene ID No. XM-027977228.2), and the 1 mutation site (the position is chr.13:7625253) existing in the No. 5 exon of the CCN5 gene is determined, the C-G mutation occurs, namely the coding sequence is mutated from the original GTA to the CTA, and the missense mutation (the coding amino acid is mutated from valine to leucine) is formed. Wherein the mutant/wild type sequence is as follows:
GACCCCAGTTTTCTGGCTTTGTCGCTCCCCCAGCCCCTGGCGTCTCCTGCCCGGAATGGAGCACCGCCTGGGGTCCCTGCTCGACCACCTGCGGCCTGGGCGTGGCCACCCGAGTGTCCAATCAGAACCGTTTCTGCCGCCTGGAGACCCAGCGCCGCCTGTGC(C/G)TACTGGGGCCCTGCCCACCCGCCAGGGGCCACGGCCCAAGGAGCAGAGCCTTTTAG
(III) analysis of polymorphism of sheep CCN5 Gene
Two results appear after Sanger sequencing through comparison, and if different genotypes appear in the overall sequence comparison result (figure 2), the results shown in figure 3 are used for checking to determine the specific sequence (genotype) of an individual. As a result, the 5 th exon of the CCN5 gene is found to generate three genotypes of CC, CG and GG in the collected small tailed Han sheep population. Among them, 36 GG genotypes, 12 CC genotypes, 48 CG genotypes and G are dominant alleles.
Further analysis showed that (Table 1), the mutation site present in exon5 of the CCN5 gene was a SNP site.
TABLE 1 frequency, genotype frequency and genetic index of CCN5 gene of small tailed Han sheep
Figure BDA0003688619060000051
Association analysis of genetic variation and meat quality traits of (IV) sheep CCN5 gene
Meat quality character data: the pH value, shearing force, flesh color, water content, tenderness and intramuscular fat content of the longissimus dorsi of the 96 small-tailed Han sheep of 6 months old are detected.
Genotype data: the genotype (CC, CG or GG) of the CCN5 gene of 96 small-tailed Han sheep with the age of 6 months.
Table 2. meat quality character difference significance test results of CCN5 gene types of small tailed han sheep
Figure BDA0003688619060000052
Note: the different shoulder mark letters represent obvious difference and have statistical significance (P is less than 0.05).
Referring to table 2, through the combined analysis of each genotype and the actual meat quality traits in the small tailed han sheep population, the intramuscular fat content of individuals with CG genotype and CC genotype in the population is obviously greater than that of individuals with GG genotype, which indicates that the meat tenderness of individuals containing C allele is obviously higher than that of homozygous wild type (GG) individuals. And the experimental and analytical results show that the SNP locus of the CCN5 gene is not obviously related to other detected meat quality traits except the meat tenderness.
Combining the above correlation analysis results, it can be presumed that: due to the fact that different amino acids are generated by mutation on the 5 th exon of the sheep CCN5 gene, the secondary structure of the protein and the conformation of the tertiary structure of the protein are changed, and therefore differences in meat quality characters, namely differences in meat tenderness, are generated by different genotypes.
In a word, the sheep CCN5 gene genetic polymorphism is analyzed by a Sanger sequencing technology and is combined with meat quality character data for correlation analysis, candidate genes which influence the sheep meat tenderness, namely CCN5 genes, are disclosed from a molecular level, and the sheep population with the dominant meat tenderness can be selected early according to polymorphic sites on the CCN5 genes, so that a theoretical basis and a scientific basis are provided for breeding of high-quality sheep.
<110> Jilin province academy of agricultural sciences
<120> detection method of genetic polymorphic site related to sheep meat tenderness and application
<160> 3
<210> 1
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 1
aacggaggga aggacatcaa at 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 2
agcggctgac atagagtttc ca 22
<210> 3
<211> 221
<212> DNA
<213> reference sequence of region near SNP site of exon5 of sheep CCN5 gene
<400> 3
gaccccagtt ttctggcttt gtcgctcccc cagcccctgg cgtctcctgc ccggaatgga 60
gcaccgcctg gggtccctgc tcgaccacct gcggcctggg cgtggccacc cgagtgtcca 120
atcagaaccg tttctgccgc ctggagaccc agcgccgcct gtgcgtactg gggccctgcc 180
cacccgccag gggccacggc ccaaggagca gagcctttta g 221

Claims (10)

1. A method for detecting single nucleotide polymorphism of sheep CCN5 gene is characterized by comprising the following steps: the method comprises the following steps:
a partial fragment of a CCN5 gene is amplified by PCR by taking sheep genome DNA or DNA contained in sheep in-vitro biological materials as a template, and the genotype of the 5 th exon single nucleotide polymorphism site of the CCN5 gene is identified by using the fragment.
2. The method for detecting the single nucleotide polymorphism of the sheep CCN5 gene according to claim 1, wherein the method comprises the following steps: the single nucleotide polymorphism site is located at chr.13:7625253, and the site has C-G mutation in sheep population.
3. The method for detecting the single nucleotide polymorphism of the sheep CCN5 gene according to claim 1, wherein the method comprises the following steps: the primers of the PCR are as follows:
an upstream primer: 5'-AACGGAGGGAAGGACATCAAAT-3'
A downstream primer: 5'-AGCGGCTGACATAGAGTTTCCA-3' are provided.
4. The method for detecting the single nucleotide polymorphism of the sheep CCN5 gene according to claim 1, wherein the method comprises the following steps: the sheep in vitro biomaterial is blood collected from sheep individuals.
5. The method for detecting the single nucleotide polymorphism of the sheep CCN5 gene according to claim 1, wherein the method comprises the following steps: the reaction system of the PCR comprises: 50-150 ng/mu L of sheep genome DNA or 1-2 mu L of sheep blood sample, and 0.4-0.5 mu L of each of 10-20 mu mol/L of upstream primer and downstream primer; the reaction conditions of the PCR include: 5min at 94 ℃; at 94-95 ℃ for 30s, at 55-65 ℃ for 30s, and at 72 ℃ for 16s, for 35 cycles; and 8min at 72 ℃.
6. The method for detecting the single nucleotide polymorphism of the sheep CCN5 gene according to claim 1, wherein the method comprises the following steps: the identification of the genotype specifically comprises the following steps: the fragments are sequenced and the sequencing result is then aligned with the reference sequence.
7. A kit for detecting single nucleotide polymorphism of sheep CCN5 gene is characterized in that: the kit comprises a site amplification primer for identifying the genotype of the 5 th exon single nucleotide polymorphism site of the CCN5 gene by a PCR-sequencing method.
8. The use of the method for detecting single nucleotide polymorphism of sheep CCN5 gene according to claim 1 in sheep molecular marker assisted selective breeding.
9. Use according to claim 8, characterized in that: the single nucleotide polymorphism site is located at chr.13:7625253, and individuals with the loci with the genotypes of CG or CC have better meat quality traits.
10. Use according to claim 9, characterized in that: the meat quality trait is selected from intramuscular fat content.
CN202210654294.4A 2022-06-10 2022-06-10 Detection method and application of genetic polymorphic site related to sheep meat tenderness Pending CN115011676A (en)

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Publication number Priority date Publication date Assignee Title
CN112118857A (en) * 2018-05-17 2020-12-22 株式会社橄榄生物科技 Pharmaceutical composition for preventing or treating retinal diseases comprising CCN5 as an active ingredient
CN112190709A (en) * 2020-10-27 2021-01-08 上海市东方医院（同济大学附属东方医院） Stromal cell protein CCN5 composition and application thereof
CN112410440A (en) * 2020-12-15 2021-02-26 吉林省农业科学院 Reagent, primer, kit and application for detecting sheep muscle fat content
CN113265471A (en) * 2021-05-28 2021-08-17 兰州大学 Method for detecting sheep FASN gene single nucleotide polymorphism and application of method in meat quality early screening

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Title
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樊红樱: "呼伦贝尔绵羊尾部脂肪组织的转录组差异表达分析", 中国博士学位论文全文数据库, 15 August 2016 (2016-08-15), pages 1 - 171 *
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