CN112176072A - Reagent, primer, kit and application for detecting intramuscular fat content of beef cattle - Google Patents
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Abstract
The invention relates to a reagent, a primer, a kit and application for detecting intramuscular fat content of beef cattle, and belongs to the technical field of molecular breeding of grassland red cattle. The invention provides an application of a reagent for detecting the haplotype of an HTR2A gene in detecting the intramuscular fat content of beef cattle. The application of the invention can provide reference for improving the quality of the grassland red beef and screening effective genetic marker genes, and finally provides basis for promoting the improvement of the quality of the beef fundamentally, improving the fat composition and producing high-grade beef.
Description
Technical Field
The invention relates to the technical field of molecular breeding of grassland red cattle, in particular to a reagent, a primer, a kit and application for detecting intramuscular fat content of beef cattle.
Background
The grassland red bull is a meat and milk dual-purpose variety cultivated in China, and has the advantages of strong adaptability, suitability for grazing, coarse feeding resistance, good meat quality and the like.
The intramuscular fat content is an important factor influencing the meat quality of grassland red cattle, and is an important index for measuring high-quality beef, and individuals with high intramuscular fat content have more obvious marbling or can form snowflake meat, and are delicious and juicy, and have good mouthfeel; if the content of fat in the muscle is too low, the meat quality is dry, hard and bland.
At present, intramuscular fat content of beef is mainly determined and analyzed by using a fat determinator, evaluation can be carried out after slaughter, and prediction in the growth process is difficult. Ultrasonic detection is also a method for detecting the intramuscular fat content of beef cattle in vivo, but the detection can be carried out only when the cattle grow to a certain age of a month, and the accuracy is not high. At present, a method for determining the intramuscular fat level of cattle by genetic linkage does not exist by utilizing a molecular genetics method.
Disclosure of Invention
The invention aims to provide a reagent, a primer, a kit and application for detecting intramuscular fat content of beef cattle. When the method is applied to cattle birth, whether the beef with high intramuscular fat content can be produced or not can be judged through the detection of haplotype combination, blind fattening is avoided, the feeding cost is saved, reference can be provided for the meat quality improvement of grassland red cattle and the screening of effective genetic marker genes, and finally, the basis is provided for promoting the improvement of the beef quality fundamentally, improving the fat composition and producing high-grade beef.
The invention provides an application of a reagent for detecting the haplotype of an HTR2A gene in detecting the intramuscular fat content of beef cattle.
Preferably, the site for detecting the haplotype of the HTR2A gene is located on exon 3 of the HTR2A gene.
Preferably, the sites for detecting the haplotypes of the HTR2A gene are 86bp and 164bp of exon 3 of the HTR2A gene.
The invention also provides a reagent for detecting the haplotype of the HTR2A gene and used for detecting the intramuscular fat content of beef cattle, and the reagent is used for detecting the mutation sites at the 86bp and the 164bp of the exon 3 of the HTR2A gene.
The invention also provides a primer for detecting the haplotype of the HTR2A gene for detecting the intramuscular fat content of beef cattle, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
The invention also provides a kit for detecting the haplotype of the HTR2A gene for detecting the intramuscular fat content of beef cattle, which comprises reagents for detecting the mutation sites at the 86bp and the 164bp of the exon 3 of the HTR2A gene.
The invention also provides a method for detecting the intramuscular fat content of beef cattle, which comprises the following steps: detecting mutation sites at 86bp and 164bp of an exon 3 of an HTR2A gene, analyzing a haplotype, and combining the haplotype which is positively correlated with the intramuscular fat content of beef cattle to be H2H 3; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G.
The invention also provides a method for breeding beef cattle with high intramuscular fat content based on the primer of the technical scheme, which comprises the following steps:
carrying out PCR amplification on a sample to be detected by using the primers in the technical scheme to obtain a PCR product;
directly carrying out Sanger sequencing on the PCR product, and counting the haplotype combination, wherein the intramuscular fat content of the beef cattle with the haplotype combination of H2H3 is higher; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G.
Preferably, each 20 μ L reaction system during amplification comprises 2 × Master Mix10 μ L, 0.5 μ L of each primer, 1 μ L of sample to be tested and the balance ddH2O。
Preferably, the reaction conditions for the amplification are: 2min at 95 ℃; 30s at 95 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃; finally the temperature was reduced to 4 ℃.
The invention provides an application of a reagent for detecting the haplotype of an HTR2A gene in detecting the intramuscular fat content of beef cattle. The invention takes grassland red cattle DNA as a template, detects the polymorphism and haplotype composition of the HTR2A gene by a PCR sequencing technology, analyzes the correlation between the haplotype and meat quality traits by single-factor variance, and finally researches the application of the reagent for detecting the HTR2A gene haplotype in detecting the intramuscular fat content of beef cattle. The invention proves that the grassland red bull HTR2A gene haplotype H2H3 has obvious positive correlation with the intramuscular fat content in the meat quality character, and provides a cattle HTR2A gene haplotype related with high intramuscular fat content, the method provided by the invention can judge and obtain the intramuscular fat content of the beef cattle by the cattle HTR2A gene haplotype, thereby providing guidance for the fattening work of the beef cattle, breeding and culturing the H2H3 haplotype beef cattle individual with high intramuscular fat content, providing reference for the meat quality improvement of the grassland red bull and the screening of effective genetic marker genes, and finally providing basis for promoting the fundamental improvement of the beef quality, improving the fat composition and producing high-grade beef. Test results show that the detection of the invention finds that the 3 rd exon in the 5 exons of the HTR2A gene has two SNPs which are strongly linked and can form different haplotypes; linkage analysis of the performance of the meat quality finds that haplotype H2H3 has obvious positive correlation with the intramuscular fat content and is a key haplotype for regulating and controlling the intramuscular fat content of the prairie red cattle; the invention researches a bovine HTR2A gene haplotype related to the intramuscular fat content of grassland red cattle, namely HTR2A gene haplotype H2H3 can be used as a judgment standard of high intramuscular fat content, the intramuscular fat content of haplotype H1H2 is 2.79% +/-1.13%, the intramuscular fat content of haplotype H1H3 is 2.87% +/-1.20%, the intramuscular fat content of haplotype H2H3 is 4.75% +/-0.12%, and the intramuscular fat content of haplotype H3H3 is 1.52% +/-0.15%. The result shows that the haplotype H2H3 of the HTR2A gene has a significant difference with the intramuscular fat trait (P < 0.05).
Drawings
FIG. 1 shows the result of agarose gel electrophoresis detection according to the present invention;
fig. 2 is a sequencing comparison result of the mutation of the 3 rd exon of the HTR2A gene provided by the present invention, wherein a is the sequencing comparison result of the mutation of the 3 rd exon g.c86g of the HTR2A gene, and B is the sequencing comparison result of the mutation of the 3 rd exon g.g164a of the HTR2A gene.
Detailed Description
The invention provides an application of a reagent for detecting the haplotype of an HTR2A gene in detecting the intramuscular fat content of beef cattle. The method judges the intramuscular fat content of the beef cattle according to the polymorphism. In the prior experiment, the grassland red bull population is taken as a research object, meat quality characters such as intramuscular fat content and the like are measured, primers are designed for HTR2A (NCBI accession number: NC-037339.1) gene, PCR amplification is carried out, after PCR amplification product glue is recovered, sequencing is carried out on a Sanger sequencing platform of Jinwei Zhi biology Limited company, SNPs are screened by NCBI-Blast, and C-G mutation and G-A mutation are found at 86bp and 164bp of exon 3 of HTR2A gene. In the present invention, the site for detecting the haplotype of the HTR2A gene is located on exon 3 of the HTR2A gene. In the invention, the original nucleotide sequence of the HTR2A gene is shown as SEQ ID NO. 3: acaacagcctgagttcacaacacaccagcatgattctagctataagttttcaaatcttgcattcaaccagagcagtcttactctacttcagtacagttgacatcaggctcctctgaagtactgaaagtatcttgtgttgccctttgctgttaactgtgccttttcccaggatgaaaactatccaagcagggtaaatttcatacgccagagaagctccaggtcattcactaatgctaaccttctgcatcccagggtaccggtggcctctgcccagcaagctctgcgctgtttggatttacctggatgtgctcttctccacggcctccatcatgcatctctgtgctatctccctggaccgctatgttgccattcagaaccccatccatcacagccggttcaactccagaactaaggcgtttctgaaaataattgctgtttggacgatatcagtgggtaagtggaacagtat, if mutation occurs, the C at 86bp of the exon 3 of the HTR2A gene is mutated into G, and the G at 164bp is mutated into A. The invention detects 2 SNP loci S1 and S2 in the 3 rd exon, the C > G mutation exists in the S1 locus at 86bp of the 3 rd exon coding region of HTR2A gene, and 3 genotypes are formed: CC. GG and CG, there are 2 alleles: c and G; the S2 site has G > A mutation at 164bp of the coding region of the 3 rd exon of the HTR2A gene, and 3 genotypes are formed: GG. AA and GA, there are 2 alleles: a and G. Haplotype analysis is carried out by utilizing HaploView software, and D' between S1 and S2 sites is 0.794, r2 is 0.350, which belongs to strong linkage. The two sites (S1, S2) form 4 haplotypes H1(CG), H2(GA), H3(GG) and H4(CA) with haplotype frequencies of 0.570, 0.234, 0.163 and 0.033 respectively.
In the invention, the sites for detecting the haplotype of the HTR2A gene are 86bp and 164bp of exon 3 of the HTR2A gene. In the invention, preferably, upstream and downstream sequences of the two SNP sites are used as templates to design genotyping primers (F and R), a PCR sequencing method is used for genotyping, and HaploView software is used for haplotype analysis. And (3) carrying out difference significance test on the intramuscular fat content corresponding to different genotypes by using SPSS 22.0 statistical software.
The invention also provides a reagent for detecting the haplotype of the HTR2A gene and used for detecting the intramuscular fat content of beef cattle, and the reagent is used for detecting the mutation sites at the 86bp and the 164bp of the exon 3 of the HTR2A gene.
The invention also provides a primer for detecting the haplotype of the HTR2A gene for detecting the intramuscular fat content of beef cattle, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2. The primer of the invention can be used for detecting two mutation sites forming a haplotype at one time. The nucleotide sequence of the upstream primer (F) is as follows: 5'-ACAACAGCCTGAGTTCACA-3' (SEQ ID NO. 1); the nucleotide sequence of the downstream primer (R) is as follows: 5'-ATACTGTTCCACTTACCCACT-3' (SEQ ID NO. 2).
The invention also provides a kit for detecting the haplotype of the HTR2A gene for detecting the intramuscular fat content of beef cattle, which comprises reagents for detecting the mutation sites at the 86bp and the 164bp of the exon 3 of the HTR2A gene.
The invention also provides a method for detecting the intramuscular fat content of beef cattle, which comprises the following steps: detecting mutation sites at 86bp and 164bp of an exon 3 of an HTR2A gene, analyzing a haplotype, and combining the haplotype which is positively correlated with the intramuscular fat content of beef cattle to be H2H 3; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G. The invention discovers that the H2H3 haplotype of the grassland red bull HTR2A gene is obviously and positively correlated with the intramuscular fat in the meat quality character. The result of the invention can provide reference basis for the meat quality improvement of grassland red cattle and the screening of effective genetic marker genes.
The invention also provides a method for breeding beef cattle with high intramuscular fat content based on the primer of the technical scheme, which comprises the following steps:
carrying out PCR amplification on a sample to be detected by using the primers in the technical scheme to obtain a PCR product;
directly carrying out Sanger sequencing on the PCR product, and counting the haplotype combination, wherein the intramuscular fat content of the beef cattle with the haplotype combination of H2H3 is higher; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G. The invention detects the genotypes of 86bp and 164bp of the exon 3 of the grassland red bull HTR2A gene by utilizing the specific primers in the technical scheme, and selects H2H3 gene haplotype grassland red bull individuals with high intramuscular fat content character correlation for cultivation.
In the present invention, the size of the PCR product is 469bp, and the PCR product is preferably sent to a sequencing company for Sanger sequencing. After the PCR amplification reaction is finished, the PCR amplification product is preferably detected by 1% agarose gel electrophoresis for about 30min, and after the detection, the gel cutting recovery is preferably carried out. After sequencing, the invention preferably utilizes DNAMAN software to analyze the sequencing result, searches SNP sites and utilizes HaploView software to analyze haplotypes.
In the invention, each 20 microliter reaction system during amplification comprises 2 xMaster Mix10 microliter, 0.5 microliter of each primer, 1 microliter of sample to be detected and the rest ddH2O。
In the present invention, the reaction conditions for the amplification are: 2min at 95 ℃; 30s at 95 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃; finally the temperature was reduced to 4 ℃.
In the invention, the sample to be detected is preferably beef cattle genome DNA, and the invention preferably extracts the genome DNA of the beef cattle sample and then stores the genome at-20 ℃.
The reagent, primer, kit and application for detecting intramuscular fat content of beef cattle are described in further detail below with reference to specific examples, and the technical scheme of the invention includes but is not limited to the following examples.
Example 1
1. Materials and methods
1) Test animal
And randomly selecting 58 beef heads of 30-month-old prairie red cattle, and slaughtering to determine meat quality characters. Collecting longissimus dorsi tissue, storing at-80 deg.C, and extracting DNA with kit at later stage.
2) Main instrument and reagent
Ultramicrospectrophotometers (Quawell-Q500), gas chromatographs (GC-14CPTF), high performance liquid chromatographs (WATERS 600), PCR instruments (T100), colorimeters (X-RitesP62), genomic DNA extraction kits and the like are all purchased from Dalibao bioengineering Co., Ltd.
3) Meat quality trait determination
The meat quality character is measured according to the interim standard of beef cattle slaughter test in China, and the meat quality character mainly comprises cooked meat rate, tenderness, water loss rate, drip loss, intramuscular fat content and marbling.
4) Genomic DNA extraction
Extracting according to a genome DNA extraction kit, determining the purity and concentration of DNA by using an ultramicro spectrophotometer, and detecting agarose gel electrophoresis by taking 5 mu l of the DNA and ensuring the integrity of the DNA.
5) Primer design and Synthesis
The gene exons were searched by Ensembl based on the DNA sequence of beef cattle HTR2A gene published by GenBank (accession No.: NC-037339.1), primers were designed using Primer 5.0, and the Primer information is shown in Table 1. Primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc.
TABLE 1 Gene primer sequences
6) PCR reaction system and conditions
PCR reaction (20. mu.l): 2 × Taq Master Mix10 μ l, ddH2O8. mu.l, DNA 1. mu.l, and upstream and downstream primers 0.5. mu.l, respectively.
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing temperature (see table 1) for 30s, extension at 72 ℃ for 30s, for 35 cycles; final extension at 72 ℃ for 5min, and storage of the product at 4 ℃.
7) Grassland red bull HTR2A gene polymorphism detection and haplotype analysis
The 58 grassland red cattle DNA samples were subjected to PCR amplification, 5. mu.l of each sample was subjected to 1.0% agarose gel electrophoresis (FIG. 1, results of agarose gel electrophoresis), and the identified products were sent to Jinzhi Biotech, Suzhou, Inc. for Sanger sequencing. And analyzing the sequencing result by using DNAMAN software to search the SNP site. Sequencing peak maps were analyzed using Chromas software. Haplotype analysis was performed using haploView software.
8) Statistical analysis of data
And (3) carrying out one-factor variance analysis on the correlation and significant difference of characters for different haplotypes and grassland red beef by using SPSS 22.0 software. Results are expressed as mean ± sd, with P <0.05 as the criterion for significance of difference.
2. Results
1) PCR sequencing results
2 SNP sites S1, S2 were detected in exon 3. FIG. 2 shows the sequencing alignment of exon 3 mutations of the HTR2A gene. Comparing the sequencing results with the reference sequence (Genbank: NC-037339.1) found that: the S1 site has C > G mutation at 86bp of the coding region of the 3 rd exon of the HTR2A gene (A in figure 2 is the sequencing comparison result of the 3 rd exon g.C86G mutation of the HTR2A gene), and 3 genotypes are formed: CC. GG and CG, there are 2 alleles: c and G. The S2 site has G > a mutation at 164bp of the coding region of exon 3 of HTR2A gene (B in fig. 2 is the sequencing alignment result of exon 3 g.g164a mutation of HTR2A gene), resulting in 3 genotypes: GG. AA and GA, there are 2 alleles: a and G.
2) Haplotype analysis
The linkage disequilibrium analysis was performed on the 2 SNPs sites detected. The analysis shows that D' between S1 and S2 is 0.794, r2 is 0.350, and the linkage is strong. The two sites (S1, S2) form 4 haplotypes H1(CG), H2(GA), H3(GG) and H4(CA) with haplotype frequencies of 0.570, 0.234, 0.163 and 0.033 respectively.
3) Correlation analysis of different haplotypes of HTR2A gene and intramuscular fat content
The correlation between different haplotypes of the HTR2A gene and the intramuscular fat character is researched by utilizing an analysis method of SPSS statistics one-way ANOVA. The result shows that the haplotype H2H3 of the HTR2A gene has a significant difference (P <0.05) with the intramuscular fat trait, and is in positive correlation. The HTR2A gene haplotype H2H3 is the judgment standard of high intramuscular fat content, and if beef cattle with high intramuscular fat content are to be bred, an individual with the H2H3 genotype can be considered to be selected.
TABLE 2 correlation analysis of different haplotypes of HTR2A gene and intramuscular fat content traits
Note: significant difference was indicated between different lower case letters (p <0.05), and very significant difference was indicated between different upper case letters (p < 0.01); the other haplotypes have low occurrence probability and cannot meet the statistical requirement, so statistics is not carried out.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. Application of a reagent for detecting the haplotype of the HTR2A gene in detecting the intramuscular fat content of beef cattle.
2. The use of claim 1, wherein the site for detecting the haplotype of the HTR2A gene is located on exon 3 of the HTR2A gene.
3. The use of claim 1 or 2, wherein the sites for detecting the haplotypes of the HTR2A gene are at the 86bp and 164bp positions of exon 3 of the HTR2A gene.
4. A reagent for detecting the haplotype of the HTR2A gene and used for detecting the intramuscular fat content of beef cattle is characterized in that the reagent is used for detecting the mutation sites at the 86bp and the 164bp of the exon 3 of the HTR2A gene.
5. A primer for detecting the haplotype of an HTR2A gene and used for detecting the intramuscular fat content of beef cattle is characterized by comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
6. A kit for detecting the haplotype of the HTR2A gene and detecting the intramuscular fat content of beef cattle is characterized by comprising reagents for detecting the mutation sites at 86bp and 164bp of exon 3 of the HTR2A gene.
7. A method for detecting intramuscular fat content of beef cattle comprises the following steps: detecting mutation sites at 86bp and 164bp of an exon 3 of an HTR2A gene, analyzing a haplotype, and combining the haplotype which is positively correlated with the intramuscular fat content of beef cattle to be H2H 3; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G.
8. A method for breeding beef cattle with high intramuscular fat content based on the primer of claim 5, which comprises the following steps:
carrying out PCR amplification on a sample to be detected by using the primer of claim 5 to obtain a PCR product;
directly carrying out Sanger sequencing on the PCR product, and counting the haplotype combination, wherein the intramuscular fat content of the beef cattle with the haplotype combination of H2H3 is higher; in the H2H3, H2 indicates that the 86bp position of the exon 3 of the HTR2A gene on one chromosome is G, the 164bp position is A, H3 indicates that the 86bp position of the exon 3 of the HTR2A gene on the other chromosome is G, and the 164bp position is G.
9. The method of claim 8, wherein the amplification reaction system comprises 10 μ L of 2 xMasterMix, 0.5 μ L of each primer, 1 μ L of the sample to be tested, and the balance ddH per 20 μ L of the reaction system2O。
10. The method of claim 8, wherein the amplification reaction conditions are: 2min at 95 ℃; 30s at 95 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; 5min at 72 ℃; finally the temperature was reduced to 4 ℃.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011053207.7A CN112176072B (en) | 2020-09-29 | 2020-09-29 | Reagent, primer, kit and application for detecting intramuscular fat content of beef cattle |
| AU2020103196A AU2020103196A4 (en) | 2020-09-29 | 2020-11-03 | Reagent, primer, and kit for detecting intramascular fat content of beef cattle, and use |
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