CN110468213B - Molecular marker related to contents of inosinic acid and intramuscular fat in golden black chicken and application of molecular marker - Google Patents
Molecular marker related to contents of inosinic acid and intramuscular fat in golden black chicken and application of molecular marker Download PDFInfo
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- CN110468213B CN110468213B CN201910699521.3A CN201910699521A CN110468213B CN 110468213 B CN110468213 B CN 110468213B CN 201910699521 A CN201910699521 A CN 201910699521A CN 110468213 B CN110468213 B CN 110468213B
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Abstract
The invention relates to the field of poultry breeding, and relates to a molecular marker related to the inosinic acid and intramuscular fat content of Jinmao black chickens and application thereof. The molecular marker is located at 27463bp of a chicken GAS6 gene and is marked as g.C27463T. The invention also discloses application of the molecular marker in breeding of the Jinmao black chickens, the method is simple and quick, is not influenced by external environment, and provides reference for molecular breeding related to the content of inosinic acid and intramuscular fat of the Jinmao black chickens.
Description
Technical Field
The invention relates to the field of poultry breeding, in particular to a molecular marker related to the contents of inosinic acid and intramuscular fat of Jinmahei chickens based on a GAS6 (i.e. growth arrest specific gene 6) gene and application thereof.
Background
At present, chicken is the second largest meat consumer product of pork in China. After the broiler chickens are genetically improved, the body weight gain, the feed conversion rate, the growth speed and the breast muscle weight are remarkably increased, and the selection process produces modern commercial chicken strains, namely the broiler chicken strains with the characteristics of high growth speed, high breast meat yield and the like. However, excessive pursuit for growth rate has resulted in broiler chicken with excessive deposition of abdominal fat and reduction of intramuscular fat. Research shows that the content of inosinic acid and intramuscular fat is closely related to the flavor and tenderness of chicken, and the quality of the chicken is influenced. At present, increasing the content of inosinic acid and intramuscular fat has become one of the main targets of broiler breeding.
The meat quality traits are complex quantitative traits and are controlled by a plurality of genes, and the traditional breeding method is difficult to make great genetic progress on the meat quality traits. SNPs are used as third-generation molecular markers, have wide application prospects in animal breeding, search and identify SNPs related to the content of inosinic acid and intramuscular fat, and are used for marker-assisted selection, so that the genetic improvement speed of the content characters of the inosinic acid and the intramuscular fat can be accelerated.
Growth arrest specific gene product 6 (GAS 6), a protein encoded by a framework protein consisting of 678 amino acids, which is a ligand for TAM receptors in the receptor tyrosine kinase family, was first discovered and cloned in NIH3T3 cells cultured in serum-free medium by Nagata et al, and has a wide range of biological effects. The research shows that the GAS6 gene is differentially expressed in the differentiation process of chicken fat precursor cells and is closely related to chicken fat deposition.
However, the technology for improving the contents of inosinic acid and intramuscular fat in the golden black chicken by using the molecular marker of the GAS6 gene is still lacked.
Disclosure of Invention
In order to overcome the defects, the technical problem to be solved by the invention is to provide a molecular marker related to the contents of the golden black chicken inosinic acid and the intramuscular fat and an application thereof, wherein the GAS6 gene molecular marker is utilized to improve the contents of the golden black chicken inosinic acid and the intramuscular fat.
In order to solve the problems, the invention discloses a molecular marker related to the inosinic acid and intramuscular fat content of black golden pheasant based on a GAS6 gene, wherein the molecular marker is positioned at the 27463bp position of a chicken GAS6 gene and is marked as g.C27463T. The chicken GAS6 gene at 27463bp is TT or CC or CT.
The invention also discloses a primer group for detecting the molecular markers related to the contents of the golden black chicken inosinic acid and the intramuscular fat, the primer group comprises a group of GAS6-P1 primer pairs,
the nucleic acid sequence of the GAS6-P1 primer pair is as follows:
an upstream primer F: 5'-GACACCTGTGCGGTCAGACT-3', as shown in SEQ ID NO. 1;
a downstream primer R: 5'-TCACTGTCTGCCACATGCCA-3', as shown in SEQ ID NO. 2.
The invention also discloses a method for detecting the molecular marker related to the contents of the golden black chicken inosinic acid and the intramuscular fat, which comprises the following steps: and (3) performing PCR amplification by using chicken genome DNA as a template and using the specific primer group to obtain a PCR product, and typing the PCR product after SSCP analysis.
The PCR amplification system consists of chicken DNA template, upstream primer F and downstream primer R, ddH2O, Taq polymerase, dNTPs, MgCl2And reaction buffer solution. Optionally, the PCR amplification system is: 2 XTaq Master Mix for Page 10. mu.L, DNA template 1. mu.L, upstream primer 0.8. mu.L, downstream primer 0.8. mu.L, ddH2O7.4 μ L; taq Master Mix for Page includes Taq polymerase, dNTPs, MgCl2And a reaction buffer solution.
Optionally, the PCR amplification procedure is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
The SSCP reaction procedure was: mixing the PCR product with the sample buffer solution, firstly denaturing, and then rapidly carrying out ice bath to enable the DNA to be in a single-stranded state; and (3) taking the denatured PCR product to perform non-denatured polyacrylamide gel electrophoresis, and then performing silver staining and color development. More specifically, the SSCP reaction program was: 2.5. mu.L of LPCR product was mixed with 7.5. mu.L of a loading buffer (98% deionized formamide, 0.025% bromophenol blue, 0.025% xylene FF, 10mmol/LEDTA (pH8.0), 2% glycerol), denatured at 98 ℃ for 10min, and then rapidly iced for 10min to make the DNA single-stranded. Taking 8 mu L of denatured PCR product, carrying out non-denaturing polyacrylamide gel electrophoresis (PAGE 30%, Acr: Bis ═ 29: 1), carrying out pre-electrophoresis at 220V for 10min, carrying out electrophoresis at 120V for 10-12h, and carrying out silver staining and color development.
The invention also discloses application of the molecular marker with the contents of the golden black chicken inosinic acid and the intramuscular fat in chicken breeding or improvement of the contents of the golden black chicken inosinic acid and the intramuscular fat.
Compared with the prior art, the invention can obtain the following technical effects:
the GAS6 gene has a mutation site at the 27463bp position, and the mutation site is obviously related to the content of inosinic acid and intramuscular fat of chicken and is easy to detect. Therefore, the invention provides a molecular marker related to the content of inosinic acid and intramuscular fat in chicken based on the GAS6 gene, a detection method of the molecular marker and application of the molecular marker in improvement of the content of inosinic acid and intramuscular fat in chicken, and provides a new method for molecular marker-assisted breeding of chicken qualitative characters.
Drawings
FIG. 1 is a gel electrophoresis diagram of the 27463bp mutation polyacrylamide of the chicken GAS6 gene, namely a PCR-SSCP electrophoresis diagram of the product of the GAS6 gene P1 (namely a PCR product obtained by amplifying a GAS6-P1 primer group);
FIG. 2 is a sequence diagram of the 27463bp mutation of the chicken GAS6 gene, namely the sequence comparison of the GAS6 genes CC, TT and CT;
FIG. 3 shows the nucleotide fragments amplified by the primer set GAS6-P1 of the present invention, the primer sequences are underlined, and the mutation sites are boxed.
Detailed Description
The following describes in detail embodiments of the present invention with reference to specific examples.
120 pieces of 112-day-old golden black chickens in the same batch of Jiangsu Sandeli animal husbandry development Limited company are collected, blood samples are collected, genome DNA is extracted, and the collected blood samples are dissolved in TE and NANODROP1000 nucleic acid concentration determinator to determine the concentration and purity and then stored at the temperature of-20 ℃ for later use.
1 pair of primers was designed using Primer Premier 5.0 based on the GAS6 gene sequence published in GenBank (accession No.: NC-006088.5). The amplification product size was 211 bp. The primer sequences are as follows:
GAS 6-P1: an upstream primer: 5'-GACACCTGTGCGGTCAGACT-3' (SEQ ID NO: 1)
A downstream primer: 5'-TCACTGTCTGCCACATGCCA-3' (SEQ ID NO: 2)
The primer group is utilized, chicken genome DNA is taken as a template for PCR amplification, and the PCR reaction system is as follows: 2 XMaster Mix for Page 10. mu.L, forward primer 0.8. mu.L, reverse primer 0.8. mu.L,DNA template μ L, ddH2O7.4. mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extension at 72 deg.C for 7min, and storage at 4 deg.C.
mu.L of the PCR product was mixed with 7.5. mu.L of a loading buffer (98% deionized formamide, 0.025% bromophenol blue, 0.025% xylene FF, 10mmol/LEDTA (pH8.0), 2% glycerol), denatured at 98 ℃ for 10min, and rapidly ice-cooled for 10min to render the DNA single-stranded. Taking 8 mu L of denatured PCR product, carrying out non-denaturing polyacrylamide gel electrophoresis (PAGE 30%, Acr: Bis: 29: 1), carrying out 220V voltage pre-electrophoresis for 10min, carrying out 120V voltage electrophoresis for 10-12h, carrying out silver staining and developing, photographing to find that three different band types exist, namely CC, TT and CT genotypes (figure 1).
3 samples of each genotype were submitted to Shanghai Biotechnology engineering services, Inc. for sequencing. The sequencing result is compared with the GAS6 gene pro sequence (GenBank ID: NC-006088.5) to find that a C → T mutation exists at 27463bp, which is marked as g.C27463T (figure 2).
The genetic variation analysis of 3 genotypes of the GAS6 gene is carried out, the allele frequency and the genotype frequency are respectively calculated, and the result shows that the C and T gene frequency is 0.39 and 0.61 respectively, the CC, CT and TT genotype frequency is 0.26, 0.26 and 0.48 respectively, the heterozygosity of the g.C27463T mutation site is 0.543, the Polymorphism Information Content (PIC) value is 0.352, and the effective allele factor is 1.84.
In order to establish the correlation between the G.C27463T mutant site of the GAS6 gene and the inosinic acid and the intramuscular fat content of the Jinmao black chickens, 120 112-day-old Jinmao black chickens are collected by Jiangsu Sandeli animal husbandry development limited company, the contents of the inosinic acid and the crude fat are slaughtered and determined, data are analyzed, the G.C27463T mutant site of the GAS6 gene and the contents of the inosinic acid and the intramuscular fat are subjected to correlation analysis by using a General Linear Model (GLM) in SPSS 22.0 software, and the results are all expressed by the mean value +/-standard deviation.
As can be seen from Table 1, the inosinic acid content of the CT genotype individuals is higher than that of the TT genotype individuals and is significantly higher than that of the CC genotype individuals (P <0.05), and the crude fat content of the CT genotype individuals is higher than that of the TT genotype individuals and is significantly higher than that of the CC genotype individuals (P < 0.01). In conclusion, the CT genotype is the dominant genotype of the invention, and in the breeding process of the Jinmao black chicken, by selecting and reserving the CT genotype individuals, the inosinic acid content and the intramuscular fat content of the Jinmao black chicken are improved, and the meat quality character can be improved.
TABLE 1 correlation analysis of GAS6 gene g.C27463T mutation and Jinmao black chicken meat quality traits
Note: the shoulder of the same row marks different lower case letters to indicate significant difference (P <0.05), and does not mark letters or the same letters to indicate no significant difference (P >0.05)
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Sequence listing
<110> Yangzhou university
<120> molecular marker related to contents of inomao black chicken inosinic acid and intramuscular fat and application
<130> xhx2019073102
<141> 2019-07-31
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gacacctgtg cggtcagact 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcactgtctg ccacatgcca 20
Claims (4)
1. The application of the primers for detecting the molecular markers related to the inosinic acid and the intramuscular crude fat content of the golden black chickens in improving the inosinic acid and the intramuscular crude fat content of the golden black chickens is characterized in that the basic group of the molecular marker at the 27463bp of a chicken GAS6 gene is C or T, the genotype at the 27463bp of a GAS6 gene is TT, CC or CT, the accession number of the GAS6 gene GenBank is NC-006088.5, and the inosinic acid content of the golden black chickens with the molecular markers of the CT genotype is higher than that of the golden black chickens with the molecular markers of TT or CC genotype; the crude fat content of the golden black chicken with the molecular marker being CT genotype is higher than that of the golden black chicken with the molecular marker being TT or CC genotype;
the nucleotide sequence of the primer is as follows:
an upstream primer F: 5'-GACACCTGTGCGGTCAGACT-3', respectively;
a downstream primer R: 5'-TCACTGTCTGCCACATGCCA-3' is added.
2. The use of claim 1, wherein the primers of claim 1 are used to perform PCR amplification using chicken genomic DNA as a template to obtain PCR products, and the PCR products are subjected to SSCP analysis and then typed.
3. The use of claim 2, wherein the PCR amplification system comprises chicken DNA template, forward primer F, and backward primer R, ddH2O, Taq polymerase, dNTPs, MgCl2And reaction buffer.
4. The use of claim 2, wherein the procedure of PCR amplification is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 61 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
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| CN101892312A (en) * | 2010-06-10 | 2010-11-24 | 扬州大学 | Molecular breeding method for chicken quality |
| CN103436630A (en) * | 2013-09-23 | 2013-12-11 | 四川省畜牧科学研究院 | High-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method |
| CN105838795A (en) * | 2016-04-27 | 2016-08-10 | 华中农业大学 | Molecular marker of related gene SVEP1 of back fat thickness and intramuscular fat traits and application of molecular marker |
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| SE0000675D0 (en) * | 2000-03-02 | 2000-03-02 | Protease Ab | Monoclonal antibodies |
| CN102858985A (en) * | 2009-07-24 | 2013-01-02 | 西格马-奥尔德里奇有限责任公司 | Method for genome editing |
| CN104673902B (en) * | 2015-02-10 | 2017-05-10 | 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 |
| CN107532333A (en) * | 2015-02-22 | 2018-01-02 | 里兰斯坦福初级大学理事会 | Microtraps screening device, method and product |
| US20170204445A1 (en) * | 2016-01-15 | 2017-07-20 | The Board Of Trustees Of The Leland Stanford Junior University | In vivo site-specific protein tagging using engineered sortase variants |
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| CN101892312A (en) * | 2010-06-10 | 2010-11-24 | 扬州大学 | Molecular breeding method for chicken quality |
| CN103436630A (en) * | 2013-09-23 | 2013-12-11 | 四川省畜牧科学研究院 | High-quality broiler chicken inosinic acid and intramuscular fat content related gene polymerization breeding method |
| CN105838795A (en) * | 2016-04-27 | 2016-08-10 | 华中农业大学 | Molecular marker of related gene SVEP1 of back fat thickness and intramuscular fat traits and application of molecular marker |
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Inventor after: Zhang Tao Inventor after: Chen Lan Inventor after: Wang Jinyu Inventor after: Xie Kaizhou Inventor after: Zhang Genxi Inventor after: Dai Guojun Inventor after: Gong Yongshuang Inventor after: Zhang Shanshan Inventor before: Zhang Tao Inventor before: Chen Lan Inventor before: Wang Jinyu Inventor before: Xie Kaizhou Inventor before: Zhang Genxi Inventor before: Dai Guojun Inventor before: Gong Yongshuang Inventor before: Zhang Shanshan |