CN111808972B - Detection method and application of correlation between GARNL1 gene and chicken testicular character - Google Patents

Detection method and application of correlation between GARNL1 gene and chicken testicular character Download PDF

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CN111808972B
CN111808972B CN202010720514.XA CN202010720514A CN111808972B CN 111808972 B CN111808972 B CN 111808972B CN 202010720514 A CN202010720514 A CN 202010720514A CN 111808972 B CN111808972 B CN 111808972B
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周敏
朱学农
谭玉文
贡继尚
许继国
文正亚
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Nanchang Normal University
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Abstract

The invention discloses a detection method and application of correlation between GARNL1 gene and testis character of chicken, belonging to the field of genetic engineering, wherein the method comprises the steps of firstly extracting the genome DNA of a chicken to be detected, then amplifying 463bp fragments at the upstream and downstream of rs14532831 locus of 5' regulatory region of GARNL1 gene, then typing the obtained PCR product by using RFLP, and then statistically analyzing the correlation between different genotypes of the gene locus rs16492031 of GARNL1 and the testis character of cocks; the invention adopts a method for detecting the correlation between the polymorphism of rs14532831 locus of the GARNL1 gene of chicken breeds and the testis characters of the chicken breeds, and uses the correlation and the method as a means for breeding and marking the chicken breeds. Compared with the existing slaughtering and breeding, the breeding method has the advantages of low cost, simple and convenient operation and accurate result.

Description

Detection method and application of correlation between GARNL1 gene and chicken testicular character
Technical Field
The invention relates to the field of genetic engineering, in particular to a method for detecting correlation between GARNL1 gene and chicken testicular character and application thereof.
Background
The GTPase-activating Rap/RanGAP domain-like 1protein (GARNL1) gene is rarely studied on chickens at present (Chen et al, 2006, 2007; Shen et al, 2012), and focuses on the gene and hen reproductive performance such as egg production, nestability, day-to-date of laying and the like. Human GARNL1 studies have shown that it is associated with neurophenotypic basal ganglia calcium carbonate (IBGC; also known as Fahr disease) (Schwarzbraun et al, 2004) and with brain developmental arrest (Shimojima et al, 2009). So far, the breeding performance of the GARNL1 gene and local chicken breeder cock 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 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. Some researches have found that the variation coefficient of local chicken testis character in the colony is as high as over 80% (Zhou Ming 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. Shen et al (2012) found that the rs14532831 site was significantly associated with the age of chicken in the day of birth (P <0.05), but whether the site was associated with the chicken testicular trait was not reported.
Disclosure of Invention
The invention aims to provide a method for detecting correlation between GARNL1 gene and chicken testicular character and application thereof, which are used for solving the problems in the prior art and using the relation and the method as a means for breeding and marking chicken breeds.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for detecting correlation between GARNL1 gene and chicken testicular character, which comprises the following steps:
(1) extracting the genome DNA of the chicken species to be detected;
(2) amplifying 463bp fragments upstream and downstream of the rs14532831 locus of the 5' regulatory region rs14532831 of the GARNL1 gene by adopting 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 GARNL1 gene locus rs14532831 and the testis traits.
Further, the primer sequence in the step (2): 5'-cctccttccacagccttccttta-3' for F and 5'-ttgcatctactgggtgggaattg-3' for R.
Further, statistical analysis is performed in the step (4) by adopting the SAS 9.0GLM program, and a model is constructed as follows: y isij＝μ+Gi+eijWherein Y isijIs the phenotypic value of a trait, μ is the overall mean of that trait, GiIs the genotype effect value, eijIs a random residual effect.
The invention also provides application of the rs14532831 genotype of the GARNL1 gene locus in detecting the testis trait correlation of chicken.
Further, the application specifically comprises the step of analyzing the relationship between the genotype and the testis character of the chicken by taking the locus rs14532831 of the chicken GARNL1 gene as a candidate marker, so as to breed the chicken variety.
Further, the chicken breeds are Ningdu yellow cocks.
The invention discloses the following technical effects:
compared with the prior art, the invention adopts a method for detecting the correlation between the polymorphism of the rs14532831 locus contained in the GARNL1 gene of the chicken variety and the testicular character of the chicken variety, and uses the relationship 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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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings 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 it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the result of the cleavage at the rs14532831 site of the GARNL1 gene; wherein: m is DS2000DNA Marker; 1.3, 6: CT type; 2. 4, 5, 8: type TT; 7: type CC.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description 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. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, 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 herein 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 present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
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 chicken GARNL1 genome sequence (GenBank accession number: JF330256) was downloaded from NCBI, and the upstream and downstream primers were designed using Genetool software, primer sequences: 5'-cctccttccacagccttccttta-3' and R5'-ttgcatctactgggtgggaattg-3', and amplifying 463bp fragments upstream and downstream of the rs14532831 site of the 5 ' regulatory region rs14532831 of the GARNL1 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 each, DNATemplate 0.6. mu.L, 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 Csp 6I, 1.0. mu.L of 10 XBuffer buffer, ddH2O2.2. mu.L, left overnight in an incubator at 37 ℃. Detecting the enzyme digestion product by 2 percent agarose gel electrophoresis. The gel imaging system photographed and the genotype was judged from the banding pattern as shown in FIG. 1.
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 isij＝μ+Gi+eij. Wherein Y isijIs the phenotypic value of a trait, μ is the overall mean of that trait, GiIs the genotype effect value, eijIs a random residual effect.
2 results and analysis
The correlation between different genotypes of the 5' regulatory region rs14532831 locus of GARNL1 gene and the testis characteristics is shown in Table 1. As can be seen from table 1, there were significant differences in testicular traits among the 6 traits tested between the different genotypes (P <0.05), with 6 traits significantly less for CC type individuals than for CT type individuals (P <0.05) and very significantly less for TT type individuals (P < 0.01).
TABLE 1 correlation of rs14532831 genotype with testis trait of caged Ningdu yellow rooster
Figure BDA0002599802730000061
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 invention uses the site rs14532831 of the 5' regulatory region of the GARNL1 gene 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 site and the testis character, so as to provide a basis for breeding and marker-assisted selection of the local chicken breeder cock.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (4)

1. A method for detecting the correlation between the GARNL1 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 463bp fragments upstream and downstream of the rs14532831 locus of the 5' regulatory region rs14532831 of the GARNL1 gene by adopting PCR reaction;
(3) carrying out RFLP typing on the obtained PCR product;
(4) performing statistical analysis on the correlation between different genotypes of the GARNL1 gene locus rs14532831 and testis traits by adopting an SAS 9.0GLM program;
the primer sequence used for PCR amplification in the step (2) is as follows: 5'-cctccttccacagccttccttta-3' for F, 5'-ttgcatctactgggtgggaattg-3' for R;
the chicken breeds are Ningdu yellow cocks;
the testicular trait is testicular weight.
2. The method of claim 1, wherein the GARNL1 gene is related to the testis trait of chicken species,
in the step (4), statistical analysis is performed by adopting an SAS 9.0GLM program, and a model is constructed as follows: y isij ＝μ+Gi +eijWherein Y isijIs the phenotypic value of a trait, μ is the overall mean of that trait, GiIs the genotype effect value, eijIs a random residual effect.
The application of a detection primer of a gene locus rs14532831 of GARNL1 in detecting the testis trait correlation of a chicken variety, wherein the chicken variety is Ningdu yellow cock; the testis character refers to the testis weight, and the sequence of the detection primer is as follows: 5'-cctccttccacagccttccttta-3' for F and 5'-ttgcatctactgggtgggaattg-3' for R.
4. The application of claim 3, wherein the application is specifically that the relationship between the genotype and the testis character of the chicken species is analyzed by taking the rs14532831 gene locus of the chicken species GARNL1 as a candidate marker, so as to breed the chicken species.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100144838A1 (en) * 2008-12-01 2010-06-10 Beck William T Methods for Identifying Modulators of Pyrimidine Tract Binding Protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100144838A1 (en) * 2008-12-01 2010-06-10 Beck William T Methods for Identifying Modulators of Pyrimidine Tract Binding Protein

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAＲNL1 基因多态位点与鸡冠高度的相关性分析;沈栩等;《仲恺农业工程学院学报》;20200630;第33卷(第2期);第11-14页 *
Laying traits and underlying transcripts,expressed in the hypothalamus and pituitary gland, that were associated with egg production variability in chickens;Chih-Feng Chen等;《Theriogenology》;20071231;第68卷(第9期);第1305-1315页 *

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