CN110878362B - Detection method for correlation between PRL gene 5' regulatory locus point and chicken testicular character and application - Google Patents
Detection method for correlation between PRL gene 5' regulatory locus point and chicken testicular character and application Download PDFInfo
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- CN110878362B CN110878362B CN201911311736.XA CN201911311736A CN110878362B CN 110878362 B CN110878362 B CN 110878362B CN 201911311736 A CN201911311736 A CN 201911311736A CN 110878362 B CN110878362 B CN 110878362B
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Abstract
The invention discloses a detection method and application of correlation between 24bp indel sites of a PRL gene 5' regulatory region and chicken testicular traits. The invention takes the 24bp indel locus of the PRL gene 5' regulatory region as a candidate marker to detect the polymorphism of the locus in the local chicken breeder Ningdu yellow cock and analyze the relation between the polymorphism and the testicular character, so as to provide a basis for breeding and marker-assisted selection of the local chicken breeder cock. 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 for correlation between a 24bp indel site of a PRL gene 5' regulatory region and a chicken testicle character and application thereof.
Background
Prolactin (PRL) is a polypeptide hormone secreted by the eosinophils of the anterior pituitary, is an important member of the growth hormone gene family, and has important regulatory effects on processes such as growth and development, endocrine and metabolism, reproduction, and immunity of the body (Bole-feepot et al, 1998, gorrin et al, 2002, jiang et al, 2006. The avian PRL polymorphism and the correlation between the polymorphism thereof and the reproductive performance of the female poultry have been studied in a large number, and particularly, the correlation between the polymorphism of the PRL gene 5' regulatory region 24bp indel site (I58724210D) and the reproductive performance of the female poultry, such as nestling behavior and the like, has been paid attention to (Jiang et al, 2005 Liang et al, 2006, xu et al, 2011. However, the correlation of the locus and the local chicken testicular character is not reported.
The testis is an important reproductive organ of a male animal and has functions of producing sperm and secreting androgen. In high-quality chicken breeding, the testicular character is not only an important economic character of laying hens, but also an important economic character of broiler breeding (Gu Minqing, 2008, orlu et al, 2009, sarabia et al, 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 finds that the variation coefficient of local chicken testicular character in the colony is more than 80% in early research (Zhou Min, and the like, 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 method takes a PRL gene 5' regulatory region 24bp indel site as a candidate marker to detect the polymorphism of the site in local chicken Ningdu yellow cock and analyze the relationship between the polymorphism and testicular character, so as to provide a basis for local chicken variety breeding and marker-assisted selection.
Disclosure of Invention
In order to overcome the defects, the invention provides a method for detecting the correlation between the polymorphism of the 24bp indel site of the PRL gene 5' regulatory region of the chicken variety and the testis shape, and the method and the relation are used as means for breeding and marking the chicken variety.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that: a detection method for correlation between 24bp indel sites of a PRL gene 5' regulatory region and chicken testicular characters is characterized by comprising the following steps:
(1) Extracting the genome DNA of the chicken species to be detected;
(2) Amplification of PRL Gene 5' regulatory region Using PCR reaction a 130bp or 154bp fragment upstream and downstream of the 24bp indel locus;
(3) Carrying out direct electrophoretic typing on the obtained PCR product;
(4) The SAS 9.0GLM program is adopted to carry out statistical analysis on the correlation between the 24bp indel locus genotype of the PRL gene 5' regulatory region and the testis traits.
Collecting 1-2 mL of blood from the infrawinged vein, 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 PRL genome sequence (GenBank accession number: NC-006089) from NCBI, and designing upstream and downstream primers by using Genetool software, wherein the primer sequences are as follows: f:5'-tttaatattggtgggtgaagagaca-3' and R:5'-atgccactgatcctcgaaaactc-3', and amplifying 130bp or 154bp fragments upstream and downstream of 24bp indel (I59724210D) site of PRL gene 5' regulatory region. The primers were synthesized by the Hunan Ongke organism Ltd.
And (3) carrying out PCR reaction system (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 20s, annealing at 54 ℃ for 20s, extension at 72 ℃ for 20s, and 35 cycles; post extension was carried out at 72 ℃ for 5min. After the reaction, all amplification products of the PRL gene were collected and subjected to 3.0% agarose gel electrophoresis to determine the genotype.
Statistical analysis is carried out in the step (4) by adopting an SAS 9.0GLM program, the model was 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.
Application of PRL gene 5' regulatory region 24bp indel locus genotype in detecting chicken testicular character correlation.
The application specifically comprises the steps of taking a 24bp indel site of a PRL gene 5' regulatory region of a chicken variety as a candidate marker, and analyzing the relationship between the genotype and the testis character of the candidate marker, thereby breeding the chicken variety.
Compared with the prior art, the invention adopts a relation for detecting the correlation between the polymorphism of the 24bpindel site of the PRL gene 5' regulatory region 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.
Drawings
FIG. 1 shows the restriction enzyme digestion result of 24bp indel site of PRL gene 5' regulatory region; wherein: m is DS2000 DNA Marker;5: a DI type; 1-4, 6-9: DD type; 10: form II.
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 flock is provided by south Jiangxi teacher science and technology Limited, the feeding time is 4 months-8 months in 2018, the number of the young chicken is 700 feathers, the feeding mode is a breeding cock cage-breeding mode, unified immunization is carried out according to a conventional immunization program of broiler breeders, and the rest is carried out feeding management according to a conventional method. The wing size was worn on the first day of birth and the 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 testis index is (testis quality/living quality) multiplied by 100, death, escape, obvious errors and repeated data are removed, and 499 test cocks are finally obtained. 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 PRL genome sequence (GenBank accession number: NC-006089) from NCBI, and designing an upstream primer and a downstream primer by using Genetool software, wherein the primer sequences are as follows: f:5'-tttaatattggtgggtgaagagaca-3', R:5'-atgccactgatcctcgaaaactc-3', amplifying a 130bp or 154bp fragment at the upstream and downstream of a 24bp indel (I59724210D) locus of a PRL gene 5' regulatory region. The primers were synthesized by the Hunan Ongke organism Ltd.
The PCR reaction system was (10. Mu.L): 2 XPCR mix 5 uL, upstream and downstream primers 0.2 uL, DNA template 0.6 uL, ddH 2 O4. Mu.L. The PCR reaction program is: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 20s, annealing at 54 ℃ for 20s, extension at 72 ℃ for 20s, and 35 cycles; post extension for 5min at 72 ℃. After the reaction, all amplification products of the PRL gene were collected and subjected to 3.0% agarose gel electrophoresis to determine the genotype.
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: y is ij ＝μ+G i +e ij . Wherein Y is ij Is the phenotypic value of a trait, μ is the overall mean of that trait, G i Is the genotype effect value, e ij Is a random residual effect.
2 results and analysis
The correlation between different genotypes of the 24bp indel (I59724210D) locus of the PRL gene 5' regulatory region and the testis traits is shown in Table 1. As can be seen from table 1, among the 6 measured traits, there were significant differences in testicular traits among different genotypes (P < 0.05), 16w total testicular mass index, 16w left testicular mass, 16w left testicular index of DD type individuals were significantly higher than those of DI type individuals (P < 0.01), 16w right testicular mass and 16w right testicular index of DD type individuals were significantly higher than those of DI type individuals (P < 0.05), and 6 indices were not significantly different from those of type II individuals.
TABLE 1 correlation of site 24bp indel (I59724210D) genotype with caged Ningdu Huang Gongji testicular trait
Based on the obtained correlation analysis results, the breeding method can be used for screening and breeding the genotype chicken species with excellent data.
The foregoing shows and describes the general principles and broad features of the present invention and 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 (4)
- A detection method for correlation between PRL gene 5' regulatory site 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 130bp or 154bp fragments upstream and downstream of a 24bp indel site of a PRL gene 5' regulatory region by adopting a PCR reaction; primer sequences for the PCR reaction:F:5’-tttaatattggtgggtgaagagaca-3’，R:5’-atgccactgatcctcgaaaactc-3’；(3) Carrying out direct electrophoretic typing on the obtained PCR product;(4) Adopting SAS 9.0GLM program to carry out statistical analysis on the correlation between different genotypes of 24bp indel sites in the PRL gene 5' regulatory region and the testis character;the chicken breeds are Ningdu Huang Gongji; the testicular trait is testicular quality;the GenBank accession number of the PRL gene is NC-006089; the PRL gene 5' regulatory region 24bp indel site is located at 59724210bp of the PRL gene.
- 2. The method for detecting correlation between PRL gene 5' regulatory locus and chicken testicular character according to claim 1, wherein the step (4) is performed by statistical analysis using SAS 9.0GLM program, and the model is constructed as follows: yij = μ + Gi + eij, where Yij is a phenotypic value of the trait, μ is an overall mean of the trait, gi is a genotypic effect value, and eij is a random residual effect.
- 3. The application of the reagent for detecting the genotype of the 24bp indel locus of the PRL gene 5 'regulatory region in the detection of the testis character correlation of the chicken is characterized in that the reagent comprises a primer sequence which can amplify 130bp or 154bp fragments upstream and downstream of the 24bp indel locus of the PRL gene 5' regulatory region; the primer sequence is F5'-tttaatattggtgggtgaagagaca-3', R5'-atgccactgatcctcgaaaactc-3';the chicken breeds are Ningdu Huang Gongji; the testicular trait is testicular quality;the GenBank accession number of the PRL gene is NC-006089; the PRL gene 5' regulatory region 24bp indel site is located at 59724210bp of the PRL gene.
- 4. The application of claim 3, wherein the application is specifically to breeding local chicken breeders cock by detecting the genotype of the 24bp indel locus of the 5' regulatory region of the chicken breeders PRL gene and analyzing the relationship between the genotype and the testicular character.
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Citations (4)
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| WO1992013102A1 (en) * | 1991-01-15 | 1992-08-06 | Genmark | Polymorphic dna markers in bovidae |
| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism |
| CN104087677A (en) * | 2014-07-22 | 2014-10-08 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for predicating milk yield of small tailed han sheep by virtue of BMPR (bone morphogenetic protein receptor )-1B or PRLR (prolactin receptor) gene SNP (single nucleotide polymorphism) lotus |
| CN105063037A (en) * | 2015-08-25 | 2015-11-18 | 新疆畜牧科学院生物技术研究所 | Hu sheep prolactin gene SNP locus and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992013102A1 (en) * | 1991-01-15 | 1992-08-06 | Genmark | Polymorphic dna markers in bovidae |
| CN101063169A (en) * | 2007-06-04 | 2007-10-31 | 西北农林科技大学 | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism |
| CN104087677A (en) * | 2014-07-22 | 2014-10-08 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | Method for predicating milk yield of small tailed han sheep by virtue of BMPR (bone morphogenetic protein receptor )-1B or PRLR (prolactin receptor) gene SNP (single nucleotide polymorphism) lotus |
| CN105063037A (en) * | 2015-08-25 | 2015-11-18 | 新疆畜牧科学院生物技术研究所 | Hu sheep prolactin gene SNP locus and application thereof |
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