CN110878363B - Detection method and application of correlation between VIPR1 gene and chicken testicular character - Google Patents
Detection method and application of correlation between VIPR1 gene and chicken testicular character Download PDFInfo
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Abstract
The invention discloses a detection method and application of correlation between VIPR1 gene locus rs15863161 genotype and chicken testicular character. The invention uses the rs15863161 site of the VIPR1 gene intron as a candidate marker to detect the polymorphism of the site in the local chicken breeder Ningdu yellow cock and analyze the relationship between the polymorphism and the testicular character, so as to provide a basis for local chicken breeder cock breeding and marker-assisted selection. Compared with the existing slaughtering and breeding, the breeding method has the advantages of low cost, simple and convenient operation and accurate result. The method is simple and easy to implement, has strong repeatability and can be carried out in a common laboratory.
Description
Technical Field
The invention relates to the field of biological genes and zoology, in particular to a detection method based on correlation of VIPR1 gene locus rs15863161 genotype and chicken testicular character and application thereof.
Background
The reproductive performance of birds is regulated by the hypothalamic-pituitary-gonadal axis, and endocrine plays a leading role in the regulation process. Experiments on in vivo and in vitro expression of vasoactive intestinal peptide type I receptor (VIPR 1) genes and correlation between VIPR1 gene polymorphism and reproductive traits prove that the VIPR1 gene is involved in the regulation and control of reproductive behaviors of female birds (Kansaku et al, 2001, you et al, 2001, zhou et al, 2008; zhouMing et al, 2011). Zenghua (2008) discovers that the VIPR1 gene has interaction relation with a plurality of genes through the interaction analysis among SNP markers, plays an important regulation role at the pituitary level in a regulation network of reproductive endocrine, and other genes mostly influence the reproductive endocrine process through the interaction with the VIPR1 gene. The avian VIPR1 polymorphism and the correlation between the polymorphism thereof and the reproductive performance of female birds have been studied in a large quantity, while the research reports on the correlation between the polymorphism and the economic traits of male birds are few (Zhao Zhenhua, etc., 2017), and the correlation with the reproductive performance of local breeding male birds has not been reported.
The testis is an important reproductive organ of a male animal and has functions of producing sperm and secreting androgen. In the breeding of high-quality chickens, the testicular character is not only an important economic character of laying hens, but also an important economic character of broiler breeding (2008 Orlu and the like, 2009 Sarabia and the like, 2013. The size and the weight of the testis of the breeding cock are directly related to the quantity and the quality of sperms and semen, which is important for the high and low fertilization rate of chicken flocks, and the breeding function of the breeding cock is gradually reduced in the late breeding stage, the quality of the semen is greatly reduced, so that the service life of the breeding cock is shortened. Meanwhile, the chicken testis as a high-quality chicken consumption byproduct has important medical and economic values, consumers in Guangdong, fujian, taiwan and the like in China have the habit of eating the chicken testis, goose testis and other poultry testis, the current market selling price can reach 100 yuan/kg, the added value of one cock testis is 2 yuan calculated according to 20g, and if the weight of the cock testis can be increased, the added value of the cock can be increased. The applicant found in early studies that the local chicken testis trait has a variation coefficient of more than 80% in the population (ZhouMin et al, 2019). And the indexes for evaluating the growth and development of the testis, such as the weight of the testis on the left side and the right side, the testis index and the total weight of the testis, are slaughter traits, if the indexes are directly selected, a large amount of slaughter is needed in field breeding and then the selection is carried out through a sibling value, and the method is high in cost and tedious in work. With the application of molecular genetic marker-assisted selection in breeding, the candidate gene method is an effective and easy-to-operate method for the chicken to carry out testicular character molecular breeding. The rs15863161 site contained in VIPR1 gene is used as a candidate marker to detect the polymorphism of the site in a local chicken breeder Ningdu yellow cock and analyze the relation between the polymorphism and the testicular character, so that a basis is provided for local chicken breeder cock breeding and marker-assisted selection.
Disclosure of Invention
In order to overcome the defects, the invention provides a method for detecting the relation between the polymorphism of the rs15863161 site in the VIPR1 gene of the chicken species and the testis shape correlation, and using the relation and the method as a means for breeding and marking the chicken species.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a detection method based on correlation of VIPR1 gene locus rs15863161 genotype and chicken testicular character is characterized by comprising the following steps:
(1) Extracting the genome DNA of the chicken species to be detected;
(2) Amplifying 434bp fragments on the upstream and downstream of VIPR1 gene locus rs15863161 by adopting a PCR reaction;
(3) Carrying out RFLP typing on the obtained PCR product;
(4) And performing statistical analysis on the correlation between different genotypes of VIPR1 gene locus rs15863161 and testis traits by adopting SAS 9.0GLM program.
Step (1), collecting 1-2 mL of blood by intravenous drip, anticoagulating with 2% EDTA, and storing at-20 deg.C for use. The blood samples were extracted with genomic DNA by conventional phenol/chloroform extraction and diluted to 100 ng/. Mu.L for use.
Step (2) downloading a chicken VIPR1 genome sequence (GenBank accession number: NM-001097523) from NCBI, and designing an upstream primer and a downstream primer by using Genetool software, wherein the primer sequences are as follows: f5 'charge ccccaaaactcagcacgac-3', R5 'charge cccaaagtccccacaaggtaa-3', amplifying 434bp fragments at the upstream and downstream of VIPR1 gene locus rs 15863161. The primers were synthesized by the Hunan Ongke organism Ltd.
And (3) performing RFLP typing on the detected PCR product. The enzyme digestion reaction system is as follows: PCR product 6.5. Mu.L, endonuclease Hha I0.3. Mu.L, 10 XBuffer buffer 1.0. Mu.L, ddH 2 O2.2. Mu.L, left overnight in an incubator at 37 ℃. Detecting the digestion product by 2% agarose gel electrophoresis. The gel imaging system takes pictures and judges the genotype according to the band type.
Performing statistical analysis by adopting an SAS 9.0GLM program in the step (4), and constructing a model as follows: yij = μ + Gi + eij. Wherein YIj is a trait phenotypic value, mu is an overall mean value of the trait, gi is a genotype effect value, and eij is a random residual effect.
Application of VIPR1 gene locus rs15863161 genotype in detection of chicken testis trait correlation.
The application specifically comprises the steps of taking a chicken species VIPR1 gene locus rs15863161 as a candidate marker, and analyzing the relationship between the genotype and the testicular character of the marker, so as to select and breed chicken species.
Compared with the prior art, the method adopts a relation for detecting the correlation between the polymorphism of the rs15863161 site contained in the VIPR1 gene of the chicken variety and the testis character, and uses the relation and the method as a means for breeding and marking the chicken variety. Compared with the existing slaughtering and breeding, the breeding method has the advantages of low cost, simple and convenient operation and accurate result. The method is simple and easy to implement, has strong repeatability and can be carried out in a common laboratory.
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FIG. 1 shows the result of enzyme digestion at site rs15863161 of VIPR1 gene; wherein: m is DS2000 DNA Marker 1: type TT; 2. 4, 5: type CC; 3: and a TC type.
Detailed Description
The following detailed description further describes the present invention for the purpose of illustrating the technical solutions and objects of the present invention.
1 materials and methods
1.1 test materials, index determination and sample Collection
The test chicken flocks are provided by south Jiangxi teacher science and technology Limited, the raising time is 4 months to 8 months in 2018, the number of the young chicken is 700 feathers, the raising mode is a breeding cock cage raising mode, unified immunization is carried out according to a conventional immunization program of broiler breeders, and the rest is subjected to raising management according to a conventional method. Wing size was worn on the first day of birth and foot size was worn on week 5. Raising to 16 weeks for slaughter. The indexes include living body mass, left testis mass, right testis mass, total testis mass and testis index. Wherein the testicular index is (testicular mass/living mass) × 100. Remove death, escape, and obvious errors and duplicate data, and finally obtain 499 test cocks. The index determination is carried out according to the method specified in NY/T823-2004 'poultry production performance noun terminology and measurement statistical method'. Collecting blood 1-2 mL by intravenous drip, anticoagulating with 2% EDTA, and storing at-20 deg.C. The blood samples were extracted with genomic DNA by conventional phenol/chloroform extraction and diluted to 100 ng/. Mu.L for use.
1.2 primer design and Synthesis
Downloading a chicken VIPR1 genome sequence (GenBank accession number: NM-001097523) from NCBI, and designing an upstream primer and a downstream primer by using Genetool software, wherein the primer sequences are as follows: f5 'ccccccgttaaactcagcagac-3', R5 'cccaaagttcccacaaggtaa-3', and a 434bp fragment upstream and downstream of VIPR1 gene locus rs15863161 is amplified. The primers were synthesized by Hainan Okangke BioLimited.
The PCR reaction system was (10. Mu.L): 2 XPCR mix 5. Mu.L, upstream and downstream primers 0.2. Mu.L each, DNA template 0.6. Mu.L, ddH 2 O4. Mu.L. The PCR reaction program is: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 58.2 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; post extension for 5min at 72 ℃. The PCR product was electrophoresed through 1% agarose gel to determine if the fragment size was as expected. The PCR products after detection were typed using RFLP. The enzyme digestion reaction system is as follows: 6.5. Mu.L of PCR product, 0.3. Mu.L of endonuclease Hha I, 1.0. Mu.L of 10 XBuffer buffer, 2.2. Mu.L of ddH2O, and left overnight in an incubator at 37 ℃. Detecting the enzyme digestion product by 2 percent agarose gel electrophoresis. The gel imaging system takes pictures and judges the genotype according to the banding pattern.
1.3 statistical analysis
As the observed groups have the same genetic background, all 16w cocks are raised under the same raising standard, the association analysis between the marker and the testicular traits adopts the SAS 9.0GLM program for statistical analysis, and the model is constructed as follows: yij = μ + Gi + eij. Wherein YIj is a trait phenotypic value, mu is an overall mean value of the trait, gi is a genotype effect value, and eij is a random residual effect.
2 results and analysis
The correlation between different genotypes of VIPR1 gene locus rs15863161 and testis traits is shown in Table 1. As can be seen from table 1, among the 6 measured traits, there is a significant difference in testicular trait (P < 0.05) between different genotypes, 6 traits of TT type individuals are significantly (P < 0.05) or significantly (P < 0.01) higher than those of CC type and TC type individuals, and 6 traits of CC type individuals and TC type individuals are not significantly different (P > 0.05).
TABLE 1 correlation of locus rs15863161 genotype with caged Ningdu yellow cock testis trait
According to the obtained correlation analysis result, the genotype chicken species with excellent data can be screened and bred.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (5)
- A detection method for correlation between VIPR1 gene and chicken testicular character is characterized by comprising the following steps:(1) Extracting the genome DNA of the chicken species to be detected;(2) Amplifying 434bp fragments on the upstream and downstream of VIPR1 gene locus rs15863161 by adopting a PCR reaction;(3) Carrying out RFLP typing on the obtained PCR product;(4) Performing statistical analysis on the correlation between different genotypes of VIPR1 gene locus rs15863161 and testis characters by adopting SAS 9.0GLM program;the chicken breeds are Ningdu yellow cocks.
- 2. The method for detecting the correlation between the VIPR1 gene and the chicken testicular character according to claim 1, wherein the primer sequence in the step (2): f:5 'ccccccgttaaactcagcagac-3', R:5 'cccaaagttcccacaaggtaa-3'.
- 3. The method for detecting the correlation between the VIPR1 gene and the testis traits of the chicken breeds according to claim 1, wherein the statistical analysis is performed by adopting SAS 9.0GLM program in the step (4), and the model is constructed as follows: yij = μ + Gi + eij, where Yij is the phenotypic value of the trait, μ is the overall mean of the trait, gi is the genotypic effect value, and eij is the random residual effect.
- The application of the rs15863161 genotype of VIPR1 gene locus in detecting the testis trait correlation of the chicken breeds is characterized in that the chicken breeds are Ningdu yellow cocks.
- 5. Use according to claim 4, characterized in that: the application specifically comprises the steps of taking a chicken species VIPR1 gene locus rs15863161 as a candidate marker, and analyzing the relationship between the genotype and the testicular character of the candidate marker, so as to breed local chicken species cocks.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104946776A (en) * | 2015-07-09 | 2015-09-30 | 马韫韬 | Chicken FABP4 gene molecular genetic marker related to good chicken slaughtering character and application of chicken FABP4 gene molecular genetic marker |
| CN110564872A (en) * | 2019-10-28 | 2019-12-13 | 东北农业大学 | Molecular marking method for predicting and identifying reproductive capacity of cocks |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104946776A (en) * | 2015-07-09 | 2015-09-30 | 马韫韬 | Chicken FABP4 gene molecular genetic marker related to good chicken slaughtering character and application of chicken FABP4 gene molecular genetic marker |
| CN110564872A (en) * | 2019-10-28 | 2019-12-13 | 东北农业大学 | Molecular marking method for predicting and identifying reproductive capacity of cocks |
Non-Patent Citations (2)
| Title |
|---|
| Identification and optimization of small molecule antagonists of vasoactive intestinal peptide receptor-1 (VIPR1);Lalgudi S Harikrishnan等;《Bioorg Med Chem Lett》;20121231;2287-90 * |
| 鸡VIPR受体基因多态性与鸡冠性状的关联分析;赵振华等;《中国家禽》;20171231;5-8 * |
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