CN114438228B - Molecular marker related to chicken muscle pH value and application thereof - Google Patents
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Abstract
The invention discloses a molecular marker related to chicken muscle pH value and application thereof, belongs to the technical field of biological genes, and provides a molecular marker related to chicken muscle pH value, wherein the molecular marker is characterized in that G+900A on exon 2 of chicken INHA gene is taken as a detection site, polymorphism of the site in local chicken Ningdu yellow cock is analyzed, and the relation between the polymorphism and pectoral muscle pH value is analyzed, and experiments show that allele A of the site is favorable for development of pectoral muscle low pH value. The method provides theoretical basis and reference data for chicken seed selection and molecular marker assisted selection through the pH value character of chicken muscles. Compared with the traditional selection and breeding through phenotype data, the detection method constructed by the molecular marker has the advantages of low cost, simple and convenient operation, accurate result, simplicity, practicability, strong repeatability and capability of being carried out in a common laboratory.
Description
Technical Field
The invention relates to the technical field of biological genes, in particular to a molecular marker related to the pH value of chicken muscle and application thereof.
Background
With the continuous upgrading of dietary structures and consumption concepts of people, the preference of chicken is gradually changed from quality to quality, and the improvement of chicken quality becomes important. Common indexes for evaluating chicken quality include meat color, flavor, water loss rate, water retention capacity, pH value and the like. Muscle traits are important economic traits for poultry and are also the main research targets for genetic improvement of poultry. The breeding of meat quality by the traditional breeding method has slow progress, and the molecular marker assisted selection method is economical, rapid and effective, and is a necessary supplement to traditional breeding. The identification of molecular markers related to meat quality traits has important scientific significance and economic value. The pH value is an index reflecting the glycolysis speed and strength of the post-slaughtered muscle of the animal and is also an important evaluation index for evaluating the quality of meat. The pH value of the muscle is 7.35-7.45 under normal physiological state, and the pH value of the muscle is 6.0-6.5 just after slaughter, and the muscle begins to rise after 1 h.
Inhibin (INH), a dimeric glycoprotein hormone secreted by ovarian granulosa cells and testis support cells, belongs to the transforming growth factor beta superfamily members, and the most fundamental biological role is to inhibit follicle stimulating hormone synthesis and secretion, and can regulate humoral follicle stimulating hormone levels through complex feedback mechanisms, and can also regulate ovarian follicle development through local action. Inhibin is composed of two different subunits, α and β, joined together by disulfide bonds, with glycosylation sites on the α units and A, B on the β units, inhibin A (αβA) and inhibin B (αβB), with neither of the isolated α and β subunits being biologically active (Ma Yongjiang et al, 2000; li Xiaoli et al, 2003). The current research of INHA genes in chickens has focused mainly on the gene polymorphism and its reproductive performance with hens, such as 300-day-old egg yield, hatchling weight, open-body quality, open-day-old, open-birth crown height, open-birth shank length, 70-day-old body weight, 98-day-old body weight/crown height, crown length, etc. (Jin Heng, 2016; gui Taotao, 2018; cui et al, 2019; fan Di, etc. 2020), and ovarian development (Lovell et al, 2003; cui et al, 2019; cui et al, 2020; wast et al, 2020; cui et al, 2021; francoeur et al, 2021). To date, the correlation between INHA gene and the pectoral muscle of breeder fowl has not been reported.
Disclosure of Invention
The invention aims to provide a molecular marker related to the pH value of chicken muscle and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a molecular marker related to the pH value of chicken muscle, and the nucleotide sequence of the molecular marker is shown as SEQ ID No. 1.
Further, there is a base mutation of A/G at the G+900A site of the sequence shown in SEQ ID No.1, and genotyping of AA, AG or GG occurs.
The invention also provides a primer pair for detecting the molecular marker related to the pH value of chicken muscle, and the nucleotide sequence of the primer pair is shown as SEQ ID No. 3-4.
The invention also provides a kit comprising the primer pair.
The invention also provides a method for detecting the molecular marker related to the pH value of chicken muscle, which comprises the following steps:
(1) Extracting genomic DNA of the chicken to be detected;
(2) Performing PCR amplification by using the primer pair to obtain an amplification product;
(3) Sequencing the amplification product obtained in the step (2) to obtain the genotype of the molecular marker;
(4) And judging the chicken pH value character of the chicken to be detected according to the genotyping result.
The invention also provides application of the molecular marker, the primer pair or the kit in detecting chicken muscle pH value characters.
The invention also provides an application of the molecular marker, the primer pair or the kit in chicken breeding.
The invention discloses the following technical effects:
the invention provides a molecular marker related to the pH value of chicken muscle, which takes G+900A on exon 2 of chicken INHA gene as a detection site, analyzes polymorphism of the site in local chicken species-Ningdu yellow cock and analyzes relationship between the polymorphism and the pH value of pectoral muscle, and finds that allele A of the site is beneficial to development of pectoral muscle with low pH value through experiments. The method provides theoretical basis and reference data for chicken seed selection and molecular marker assisted selection through the pH value character of chicken muscles. Compared with the traditional selection and breeding through phenotype data, the detection method constructed by the molecular marker has the advantages of low cost, simple and convenient operation, accurate result, simplicity, practicability, strong repeatability and capability of being carried out in a common laboratory.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the detection result of INHA gene PCR products, wherein M is DL2000 marker, and 1 is PCR product;
FIG. 2 shows the results of INHA gene sequencing;
FIG. 3 is a graph showing the results of an INHA gene sequence alignment at NCBI;
FIG. 4 shows the results of an INHA gene sequence alignment in UCSC chicken genomes;
FIG. 5 is a position of a G+900A site in exon 2 of the INHA gene, wherein the bolded letters represent the G+900A site, bolded "ATG" and "TAG" represent the start codon and stop codon, respectively, uppercase letters represent exons, lowercase letters represent introns;
FIG. 6 is a sequencing diagram of the different genotypes of the G+900A locus of the INHA gene, wherein the shaded portion is the G+900A locus.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1
1 materials and methods
1.1 test Material, index determination and sample acquisition
The test chicken flock is provided by Jiangxi southern Engineer science and technology Co-Ltd, the raising time is 4 months in 2018-8 months, the number of chicken fries is 700 feathers, the raising mode is a breeding cock cage raising mode, unified immunization is carried out according to the conventional immunization program of the broiler breeder, and the other breeder breeds management is carried out according to the conventional method. The first day of birth was fitted with a fin number and the 5 th week was fitted with a foot number. Raising to 16 weeks of age for slaughtering, correcting the pH meter with standard solution with pH value of 9.18 and pH value of 6.86, inserting probe of the acidometer into pectoral muscle of each chicken within 45min after slaughtering, ensuring that the probe is buried in muscle, recording reading of the pH meter when the reading of the pH meter is stable, and repeating for 3 times to calculate average value. Death, escape, and obvious errors and duplicate data were removed, resulting in 497 test cocks. Index determination is carried out according to the method specified in NY/T823-2004, the term for poultry production Performance and metric statistical method. Blood is collected by vein under the wing for 1-2 mL, anticoagulated by EDTA with concentration of 2 percent, and preserved at the temperature of minus 20 ℃ for standby. The collected samples were extracted with a conventional phenol/chloroform extraction method for genomic DNA and diluted to 100 ng/. Mu.L for use.
1.2 primer design and Synthesis
The chicken INHA genomic sequence (GenBank accession number: NM-001031257.2) was downloaded from NCBI, and the upstream and downstream primers were designed using Genetool software, primer sequences: f (SEQ ID No. 3): 5'-AGCTCCTGTGTC TCCCCCCATAC-3', R (SEQ ID No. 4): 5'-GTGCCATGCCAGCTGCTAGAC-3', amplified 821bp fragment upstream and downstream of the INHA gene locus. Primers were synthesized by Hunan Optimus, inc.
The PCR reaction system was (50. Mu.L): 2 XPCR mix 25. Mu.L, upstream and downstream primers each 0.4. Mu.L, DNA template 1.0. Mu.L, ddH 2 O23.2. Mu.L. The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 45s,35 cycles; and then extending at 72 ℃ for 10min. The PCR products were checked for fragment size by 1% agarose gel electrophoresis. The PCR products after detection are sent to Hunan qing Ke biological limited company for direct sequencing by using an upstream primer, and the genotype is judged according to each individual sequencing result.
1.3 statistical analysis
Because the observed groups have the same genetic background and are all 16w cock, and are bred under the condition of the same breeding standard, the association analysis between the markers and testicle characters adopts the SAS 9.0GLM program for statistical analysis, and a model is constructed as follows: y is Y ij ＝μ+G i +e ij . Wherein Y is ij Is the character phenotype value, mu is the overall average value of the character, G i Is the genotype effect value, e ij Is a random residual effect.
2 results and analysis
2.1PCR product detection results
The INHA gene PCR product was detected by 1.0% agarose gel electrophoresis as shown in FIG. 1, and there was a clear single band at 821bp consistent with the expected fragment size. The PCR product after detection is directly sequenced, the sequenced sequence is shown in figure 2, and the part with poor sequencing results of the 5 'end and the 3' end is removed, so that 760bp sequence is obtained. The obtained sequence is subjected to https:// blast.ncbi.nfm.nih.gov/blast.cgi comparison, whether the obtained sequence is the sequence of the INHA gene or not is further verified, the comparison result is shown in figure 3, and the sequence obtained by sequencing is a partial sequence of the INHA gene. The sequences obtained by sequencing were further aligned at http:// genome. Ucsc. Edu/cgi-bin/hgBlat and the results are shown in FIG. 4.
SEQ ID No.1：
ACCCTGAGCCACTCTTTCCCCACAGACGTGCCGTGCGAGCCCACGCAGCCAGACAAGCTGCTGGAGGAAGAAGGCATCTTCACTTACCTCTTCCAGCCCTCGGCGCACGCCCTGAGCCGCACGCTGACATCCGCCCAGCTCTGGTTCTACAGCGGCCCCTCGGCTGCTCCCAACCACTCGGCCCCCGCTGTGCTGACCCTCTCACCGCAGGGCAGGGTGCCGGTGGTGGCCACAGCGTCGCGGACGCCGGAGCACTGGACCGTGTTTGACTTCGGCCCCGATGCGCTGCCCCAGCTGGCACAGCCGCTCTTCGTGCTCCTGGTGCGCTGCCCCGGCTGCCCCTGCCTGGCCGATGGGGACAAGATGCCCTTCCTGGTGGCCACTACCCGTGCCAAGGCAGCCGGGAGGGCTCGCCGCTCCGCCGTGCCCTGGTCGCCGGCTGCGCTCAGCCTGCTGCAGCGCCCATCGGAGGACGTGGCCGCCCACACCAACTGCCGCCGGGCCTCCCTCAACATCTCTTTCGAGGAGCTGGGCTGGGACAATTGGATCGTGCACCCCAGCAGCTTCGTTTTCCACTACTGCCACGGGAACTGTGCCGAAGGCCACGGGCTGAGCCACCGGCTGGGGGTGCAGCTGTGCTGCGCCGCGCTGCCCGGCACCATGCGCTCGCTGCGTGTCCGCACCACCTCTGATGGTGGCTACTCCTTCAAGTACGAGACGGTGCCCAACATCCTGGCGCAGGACTGCACCTGTGTCTAGC
2.2 genotyping of the G+900A locus of the INHA Gene
497 and Huang Gongji INHA gene exon 2, and the specific position of the locus at exon 2 is shown in FIG. 5. Comparing sequencing results found that there were mutations of G > A at this site and 3 genotypes (FIG. 6).
SEQ ID No.2：
Note that: the bolded letters represent the g+900A site, bolded "ATG" and "TAG" represent the start codon and stop codon, respectively, the uppercase letters represent exons, and the lowercase letters represent introns.
2.3 correlation of INHA Gene G+900A site with the pH value of the pectoral muscle of Ningdu Huang Gongji
Correlation of the different genotypes of INHA gene locus G+900A with the pH of the pectoral muscle of Ningdu Huang Gongji at 16 weeks of age is shown in Table 1. As can be seen from table 1, this site is very significantly correlated with pectoral pH (P < 0.01); and the 6 week-old pectoral muscle pH value of the locus has extremely obvious difference (P < 0.01), the pectoral muscle pH value of the AA type individual is extremely obvious (P < 0.01) lower than that of the GG type individual, and the pectoral muscle pH value of the AG type individual is not obvious (P > 0.05) with the AA type and GG type individual.
TABLE 1 correlation of the G+900A genotype at position G and the pH of the pectoral muscle of Ningdu Huang Gongji
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Sequence listing
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acgctgacat ccgcccagct ctggttctac agcggcccct cggctgctcc caaccactcg 180
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Claims (3)
1. A method for detecting the pH value character of chicken muscle, comprising the steps of:
(1) Extracting genomic DNA of the chicken to be detected;
(2) Performing PCR amplification by using the primer pair to obtain an amplification product; the nucleotide sequence of the primer pair is shown as SEQ ID No. 3-4;
(3) Sequencing the amplification product obtained in the step (2) to obtain the genotype of the molecular marker; the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, A/G mutation exists at 669bp of the molecular marker, and genotyping of AA, AG and GG occurs;
(4) Judging the pH value character of the muscle of the chicken to be detected according to the genotyping result, wherein the pH value of the muscle of the AA genotype individual is lower than AG and GG genotypes;
the pH value of the muscle is the pH value of chest muscle of 16 weeks of age;
the chicken is a Ningdu yellow cock.
2. The application of the molecular marker in detecting the pH value character of chicken muscle is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, A/G mutation exists at 669bp of the molecular marker, and genotyping of AA, AG and GG occurs; the pH value of the muscle is the pH value of chest muscle of 16 weeks of age; the chicken is a Ningdu yellow cock.
3. The application of the molecular marker in chicken breeding is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID No.1, A/G mutation exists at 669bp of the molecular marker, genotyping of AA, AG and GG occurs, and the pH value of the muscle of an AA genotype individual is lower than that of AG and GG genotypes; the pH value of the muscle is the pH value of chest muscle of 16 weeks of age; the chicken is a Ningdu yellow cock.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673902A (en) * | 2015-02-10 | 2015-06-03 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to breast muscle weight and breast muscle percentage of chicken and application of SNP molecular marker |
| CN112322753A (en) * | 2020-11-27 | 2021-02-05 | 广西扬翔股份有限公司 | SNP molecular marker related to pork intramuscular fat and application thereof |
| CN113430284A (en) * | 2021-08-19 | 2021-09-24 | 南昌师范学院 | Molecular marker related to chicken pectoral muscle mass and pectoral muscle rate, kit and application |
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2022
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673902A (en) * | 2015-02-10 | 2015-06-03 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker related to breast muscle weight and breast muscle percentage of chicken and application of SNP molecular marker |
| CN112322753A (en) * | 2020-11-27 | 2021-02-05 | 广西扬翔股份有限公司 | SNP molecular marker related to pork intramuscular fat and application thereof |
| CN113430284A (en) * | 2021-08-19 | 2021-09-24 | 南昌师范学院 | Molecular marker related to chicken pectoral muscle mass and pectoral muscle rate, kit and application |
Non-Patent Citations (2)
| Title |
|---|
| 肌肉生长抑制素基因(MSTN)外显子1的多态性及其与边鸡生长性状的关联分析;张跟喜;丁馥香;是燕萍;王金玉;张李俊;张丽;施会强;;农业生物技术学报(01);全文 * |
| 赤水乌骨鸡肌肉生长抑制素基因多态性及其与肉质性状的关联分析;李维;祖盘玉;李洪林;林家栋;张福平;;中国畜牧兽医(04);全文 * |
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