CN110872612B - Detection method for correlation between VIPR2 gene 3' regulatory locus point and chicken testicular character and application - Google Patents

Detection method for correlation between VIPR2 gene 3' regulatory locus point and chicken testicular character and application Download PDF

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CN110872612B
CN110872612B CN201911313405.XA CN201911313405A CN110872612B CN 110872612 B CN110872612 B CN 110872612B CN 201911313405 A CN201911313405 A CN 201911313405A CN 110872612 B CN110872612 B CN 110872612B
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周敏
谭玉文
朱学农
张玉涛
陈佳坤
张盼
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Abstract

The invention discloses a detection method and application of a VIPR2 gene 3' regulatory locus point and chicken testicular character correlation. The invention uses VIPR2 gene 3' regulatory region T9699253G site as candidate marker to detect the polymorphism of the site in local chicken Ningdu yellow cock and analyze the relation between the site and testis character, so as to provide basis for local chicken variety 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

Detection method for correlation between VIPR2 gene 3' regulatory locus point and chicken testicular character and application
Technical Field
The invention relates to the field of biological genes and zoology, in particular to a detection method for correlation between a VIPR2 gene 3' regulatory region T9699253G locus and chicken testicular characters and application thereof.
Background
The vasoactive intestinal peptide type 2 receptor (VIPR 2) is a 7-fold transmembrane glycoprotein coupled to Gs protein, promotes the production of intracellular cAMP by coupling to adenylate cyclase, belongs to the secretin/VIP receptor subfamily (being the G protein coupled receptor family B subfamily), and is one of the co-receptors for Pituitary Adenylate Cyclase Activating Peptide (PACAP) and Vasoactive Intestinal Peptide (VIP) (harma, 2001; Wang et al, 2010). In studies on mammals and fish, PACAP and VIP are found not only in the central nervous system but also in peripheral tissues including the ovary, and act as important autocrine/paracrine factors in cooperation with local regulatory gonadotropins in the ovary (Park et al, 2003; Barberi et al, 2007), and in cooperation with gonadotropins involved in follicular development (Gras et al, 2005; Zhou et al, 2011), apoptosis (Flaws et al, 1995; Vaccari et al, 2006), and oocyte maturation (zhao et al, 2005; Zhou et al, 2011), both of which cause a series of biological reactions by binding to specific receptors distributed on gonadal target cells (Harmar et al, 2001; Wang et al, 2010). The chicken VIPR2 is located on chromosome 2, has a full length of 50747bp, and consists of 13 exons and 12 introns. Studies of VIPR2 in mammals and fish have shown that binding to PACAP and VIP plays an important regulatory role in the circulatory, immune, reproductive and digestive systems of animals (Vaudry et al, 2000; Harmar et al, 2012). The reports of the reproductive performance of the poultry VIPR2 and the breeding performance of the poultry are very few (Zhao Zhenhua et al, 2017), and the reproductive performance of the poultry and local chicken breeders is not reported yet.
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 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 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 VIPR2 gene 3' regulatory region T9699253G site is used as a candidate marker to detect the polymorphism of the site in local chicken Ningdu yellow cock and analyze the relation between the polymorphism and the testis character, so that a basis is provided 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 3' regulatory region T9699253G locus of the chicken VIPR2 gene and the testis shape, and using the relationship and the method as a 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 based on the correlation between the VIPR2 gene 3' regulatory region T9699253G locus and the chicken testicle character is characterized by comprising the following steps:
(1) extracting the genome DNA of the chicken species to be detected;
(2) amplifying 434bp fragments upstream and downstream of a T9699253G locus in a 3' regulatory region of the VIPR2 gene by adopting a PCR reaction;
(3) carrying out RFLP typing on the obtained PCR product;
(4) the SAS 9.0GLM program is adopted to carry out statistical analysis on the correlation between different genotypes of the T9699253G locus in the 3' regulatory region of the VIPR2 gene and the testis traits.
Step (1), collecting 1-2 mL of blood by using a parafin vein, anticoagulating with 2% EDTA, and storing at-20 ℃ for later 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 VIPR2 genome sequence (GenBank accession number: NM-001014970) from NCBI, and designing an upstream primer and a downstream primer by using Genetool software, wherein the primer sequences are as follows: 5'-ccccatttagtagccagtc-3' and 5'-gggcaggtgtctttgttat-3', and amplifying 420bp fragments upstream and downstream of the 3 ' regulatory region of the VIPR-2 gene. The primers were synthesized by Hainan Okangke BioLimited.
And (3) performing RFLP typing on the detected PCR product. The enzyme digestion reaction system is as follows: the PCR product was 6.5. mu.L,6.5. mu.L of PCR product, 0.3. mu.L of endonuclease BseG I, 1.0. mu.L of 10 XBuffer buffer, ddH2O2.2. mu.L, placed overnight in an incubator at 55 ℃. Detecting the enzyme digestion product by 3 percent agarose gel electrophoresis. The gel imaging system takes pictures and judges the genotype according to the banding pattern.
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.
The application of the VIPR2 gene 3' regulatory region T9699253G locus in detecting the testis trait correlation of chicken breeds.
The application specifically comprises the steps of analyzing the relationship between the genotype and the testis character of the chicken by taking the T9699253G locus of the 3' regulatory region of the chicken VIPR2 gene as a candidate marker, and breeding the chicken variety.
Compared with the prior art, the invention adopts a relation for detecting the correlation between the polymorphism of the T9699253G locus of the 3' regulatory region of the chicken species VIPR2 gene and the testis shape, and uses the relation and the method as a means for breeding and marking the chicken species. 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 cleavage at site T9699253G of VIPR2 gene; wherein: m is DS2000 DNA Marker 1: type TT; 2. 3, 4, 5: GG type; 6: TG 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 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 index measurement comprises 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 measurement is carried out according to the method specified in NY/T823-2004 "poultry Performance noun terminology and metrics statistics method". Collecting blood 1-2 mL in a infrawing vein, anticoagulating with 2% EDTA, and storing at-20 ℃ for later use. 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
The genomic sequence of chicken VIPR2 (GenBank accession No.: NM-001014970) was downloaded from NCBI, and the upstream and downstream primers were designed using Genetool software, primer sequences: 5'-ccccatttagtagccagtc-3' and 5'-gggcaggtgtctttgttat-3', and amplifying 420bp fragments upstream and downstream of the 3 ' regulatory region of the VIPR-2 gene. The primers were synthesized by Hainan Okangke BioLimited.
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, ddH2O4. mu.L. The PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; post extension was carried out at 72 ℃ for 5 min. The PCR product was electrophoresed through 1% agarose gel to determine if the fragment size was as expected. And typing the PCR product after detection by using RFLP. The enzyme digestion reaction system is as follows: 6.5. mu.L of PCR product, 0.3. mu.L of endonuclease BseG I, 1.0. mu.L of 10 XBuffer buffer, ddH2O2.2. mu.L, placed overnight in an incubator at 55 ℃. Detecting the enzyme digestion product by 3 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 the T9699253G locus of the 3' regulatory region of the VIPR2 gene and the testis traits is shown in Table 1. As can be seen from table 1, there were significant differences in testicular traits among the 6 traits tested (P <0.05), 6 traits significantly higher for TT-type individuals than those for GG-type individuals (P <0.05), and no significant differences in 6 traits from those for TG-type individuals (P > 0.05).
TABLE 1 correlation of the T9699253G genotype at site with the testis trait of caged Ningdu yellow rooster
Figure BDA0002325154610000041
Figure BDA0002325154610000051
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)

  1. The method for detecting the correlation between the 3' regulatory site of VIPR2 gene and the testis character of chicken breeds is characterized by comprising the following steps:
    (1) extracting the genome DNA of the chicken species to be detected;
    (2) amplifying 420bp fragments on the upstream and downstream of a T9699253G locus in a 3' regulatory region of the VIPR2 gene by adopting a PCR reaction;
    (3) performing RFLP typing on the obtained PCR product, wherein the endonuclease used for the RFLP typing is endonuclease BseG I;
    (4) adopting SAS 9.0GLM program to carry out statistical analysis on the correlation between different genotypes of T9699253G locus of 3' regulatory region of VIPR2 gene and testis characters;
    the primer sequence used for PCR amplification in the step (2) is as follows: 5'-ccccatttagtagccagtc-3' for F, 5'-gggcaggtgtctttgttat-3' for R;
    the locus T9699253G of the 3 'regulatory region of the VIPR2 gene is the 361 st locus of the PCR product in the step (3), and when the locus T9699253G of the 3' regulatory region of the VIPR2 gene is mutated from T to G, the 361 st to 365 th loci of the PCR amplification product can be recognized by endonuclease BseG I.
  2. 2. The method for detecting the correlation between the 3' regulatory locus of VIPR2 gene and the testis characteristics of chicken breeds as claimed in claim 1, wherein the statistical analysis is performed by using SAS 9.0GLM program in step (4), 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.
  3. 3. The application of a detection reagent for detecting the T9699253G locus of the 3' regulatory region of the VIPR2 gene of claim 1 in preparing a kit for detecting the testis trait correlation of chicken breeds.
  4. 4. Use according to claim 3, characterized in that: the application specifically comprises the steps of analyzing the relationship between the genotype and the testis character of the chicken by taking the T9699253G locus of the 3' regulatory region of the chicken VIPR2 gene as a candidate marker, and breeding the chicken variety.
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